Cold-inducible and tuber-specific promoter sequence from potato α-amylase gene

ABSTRACT

A cold-inducible promoter region and a tuber-specfic promoter region from an α-amylase gene from  Solanum tuberosum  are described. The promoter regions can be used to express a gene of interest in plants or plant cells.

This is the U.S. national phase under 35 U.S.C. § 371 of InternationalApplication PCT/EP95/02196, filed Jun. 6, 1995.

FIELD OF THE INVENTION

The present invention relates to a promoter, including a construct andan expression vector comprising the same and a transformed cellcomprising the same. In addition the present invention relates to aplant comprising the same.

BACKGROUND OF THE INVENTION

It is known that it is desirable to direct expression of a gene ofinterest (“GOI”) in certain tissues of an organism—such as a plant. Forexample, it may be desirable to produce crop protein products with anoptimised amino acid composition and so increase the nutritive value ofthe crop. It may even be desirable to use the crop to express non-plantgenes such as genes for mammalian products. Examples of the latterproducts include interferons, insulin, blood factors and plasminogenactivators.

However, whilst it may be desirable to achieve expression of a GOI incertain tissues it is sometimes important (if not necessary) to ensurethat the GOI is not expressed in other tissues in such a manner thatdetrimental effects may occur. Moreover, it is important not to upsetthe normal metabolism of the organism to such an extent that detrimentaleffects occur. For example, a disturbance in the normal metabolism in aplant's leaf or root tip could lead to stunted growth of the plant.

CA-A-2006454 describes a DNA sequence of an expression cassette in whichthe potato tuber specific regulatory regions are localised. Theexpression cassette contains a patatin-gene with a patatin-genepromoter. The DNA sequence is transferred into a plant genome usingagrobacteria. According to CA-A-2006454, the DNA sequence enablesheterologous products to be prepared in crops.

One of the key plant enzymes is α-amylase. α-amylase participates in thepathway responsible for the breakdown of starch to reducing sugars inpotato tubers. Genes coding for α-amylase in potato plants have beenisolated and characterised. For example, see the teachings inEP-B-0470145.

In brief, α-amylase is encoded by a gene family consisting of at least 5individual genes. Based on their homology the genes can be divided intotwo subfamilies—one of which is the type 3 amylase(s), the other ofwhich is the type 1 amylase(s). The two groups of α-amylases areexpressed differently, not only on the molecular level but also indifferent tissues of the potato plant.

In this regard, type 3 α-amylases are expressed in root, in tubers, insprouts and in stem tissue; whereas type 1 α-amylases are expressed insprout and stem tissues, but not in tubers.

SUMMARY OF THE INVENTION

The present invention seeks to provide a plant promoter that is capableof directing the expression of a gene of interest in specific tissues,or in just a specific tissue, of an organism, typically a plant.

According to a first aspect of the present invention there is provided apromoter comprising a nucleotide sequence corresponding to the 5.5 KbEcoR1 fragment isolated from Solanum tuberosum, or a variant, homologueor fragment thereof.

A restriction map of the 5.5 Kb EcoR1 fragment isolated from Solanumtuberosum is shown in FIGS. 1, 2 and 8—which are discussed later.

According to a second aspect of the present invention there is provideda promoter comprising a nucleotide sequence corresponding to the 5.5 KbEcoR1 fragment isolated from Solanum tuberosum, or a variant, homologueor fragment thereof but wherein at least a part of the promoter isinactivated.

According to a third aspect of the present invention there is provided apromoter comprising at least the nucleotide sequence shown as SEQ IDNO:1or a variant, homologue or fragment thereof.

According to a fourth aspect of the present invention there is provideda promoter comprising the nucleotide sequence of any of one of thesequences shown as Seq.I.D.No.s 4-17, preferably any of one of thesequences shown as Seq.I.D.No.s 4-16, or a variant, homologue orfragment thereof.

According to a fifth aspect of the present invention there is provided apromoter comprising a nucleotide sequence corresponding to the 5.5. KbEcoR1 fragment isolated from Solanum tuberosum, or a variant, homologueor fragment thereof, but wherein at least the nucleotide sequence shownas SEQ ID NO:1 is inactivated.

According to a sixth aspect of the present invention there is provided apromoter comprising a nucleotide sequence corresponding to the 5.5. KbEcoR1 fragment isolated from Solanum tuberosum, or a variant, homologueor fragment thereof, but wherein at least any of one of the sequencesshown as SEQ ID NOS:2-16 is inactivated.

According to a seventh aspect of the present invention there is provideda construct comprising the promoter according to the present inventionfused to a GOI.

According to an eighth aspect of the present invention there is providedan expression vector comprising the promoter according to the presentinvention.

According to a ninth aspect of the present invention there is provided atransformation vector comprising the promoter according to the presentinvention.

According to a tenth aspect of the present invention there is provided atransformed cell comprising the promoter according to the presentinvention.

According to an eleventh aspect of the present invention there isprovided a transgenic organism comprising the promoter according to thepresent invention.

According to a twelfth aspect of the present invention there is providedthe use of the promoter according to the present invention as a coldinducible promoter.

According to a thirteenth aspect of the present invention there isprovided a construct comprising the promoter of the present inventionand a nucleotide sequence coding for anti-sense alphα-amylase.

According to a fourteenth aspect of the present invention there isprovided the use of a promoter according to the present invention forexpressing a GOI in tuber and/or sprout and/or root and/or stem of aplant, preferably in just or at least tuber of a plant.

Other aspects of the present invention include methods of expressing ortransforming any one of the expression vector, the transformationvector, the transformed cell, including in situ expression within thetransgenic organism, as well as the products thereof. Additional aspectsof the present invention include uses of the promoters for expressingGOIs in vitro (e.g. in culture media such as a broth) and in vivo (e.g.in a transgenic organism).

Preferably, in any one of the expression vector, the transformationvector, the transformed cell or the transgenic organism the promoter ispresent in combination with at least one GOI.

Preferably the transformation vector is derived from agrobacterium.

Preferably the promoter is stably incorporated within the transgenicorganism's genome.

Preferably the transgenic organism is a plant. Preferably the plant is adicot plant.

More preferably, the plant is a potato plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction enzyme map;

FIG. 2 shows a restriction enzyme map;

FIG. 3 shows a nucleotide sequence of a promoter according to thepresent invention;

FIG. 4 is a pictorial representation of some deletions made to thesequence of FIG. 2;

FIG. 5 is a pictorial representation of some deletions made to thesequence of FIG. 2;

FIG. 6 shows a series of primer sequences;

FIG. 7 shows a map of pBlueScript KS (2.96 kb) and a map of pBlueScriptM13 (3.2 kb).

FIG. 8 is a restriction enzyme map.

FIG. 9 is a restriction map of pJK4; and

FIG. 10 is a map of pEPL.

DETAILED DESCRIPTION OF THE INVENTION

An advantage of the present invention is that a promoter correspondingto the 5.5. Kb EcoR1 fragment isolated from Solanum tuberosum is able todirect expression of a GOI in any one of root, tuber, sprout and stemtissue of a dicot, for example, a potato. The same is true for thevariant, homologue or fragment thereof.

Further surprising however is the fact that at least a part of thepromoter sequence can be inactivated (e.g. truncated) and it can stillexpress a GOI.

More surprising is the fact that the partially inactivated (e.g.truncated) promoter sequences can direct expression of a GOI in one ormore specific tissues, such as just tuber tissue, rather than incombination of root, tuber, sprout and stem tissues.

In this regard, it was found that modified promoters corresponding tothe 5.5 Kb EcoR1 fragment isolated from Solanum tuberosum containinginactivated nucleotide sequences upstream (i.e. towards the 5′ end) ofposition −691 with reference to FIG. 3 do not yield expression in anyone of root, tuber, sprout or stem tissue. Examples of such modifiedpromoters include modified promoters containing only nucleotidesequences downstream of position −692, such as the promoter sequencesSEQ ID NOS:2-3.

However, it was found that promoters corresponding to the 5.5 Kb EcoR1fragment isolated from Solanum tuberosum containing inactivatednucleotide sequences upstream of position −1535 (with reference to FIG.3) yield expression only in tuber tissue. Examples of these types ofpromoters include those that contain only nucleotide sequencesdownstream of position −1535 but wherein they contain at leastnucleotide sequences upstream of −691 (with reference to FIG. 3), suchas the promoter sequences SEQ ID NOS:4-17, in particular SEQ IDNOS:6-17, more in particular SEQ ID NOS:6-16.

Moreover, it was found with the last type of promoters that if thosepromoters contained at least SEQ ID NO:1 high expression yields wereobserved in tuber tissue.

Thus preferred examples of promoter sequences for tuber specificexpression of a GOI containing at least the sequence shown as SEQ IDNO:1 include those sequences shown as Seq. I.D. No.s 4-17, morepreferably those sequences shown as SEQ ID NOS:6-17, even morepreferably those sequences shown as SEQ ID NOS:6-16.

Furthermore, it was found that promoters corresponding to the 5.5 KbEcoR1 fragment isolated from Solanum tuberosum containing inactivatednucleotide sequences downstream of position −1535 (with reference toFIG. 3) yield expression in root and/or sprout and/or stem tissue.Examples of these types of promoters include those that contain onlynucleotide sequences upstream of position −1535 (with reference to FIG.3).

Moreover, it was found that promoters corresponding to the 5.5 Kb EcoR1fragment isolated from Solanum tuberosum containing an inactive SEQ IDNO:1 yield expression only in root and/or sprout and/or stem tissue.Examples of these types of promoters include those that do not containSeq. I.D. No. 1.

Particularly preferred sequences are SEQ ID NOS:4-16.

Tissue specific expression, such as tuber specific expression, isparticularly advantageous for a number of reasons.

First, a GOI (as defined below) can be expressed in a specific tissuetype. This is particularly advantageous if the GOI is an anti-senseendogenous for the organism in question because expression of theanti-sense sequence in other tissues can be detrimental.

Second, it is possible to express a GOI coding for an agent giving theorganism resistance against a disease associated with specifictissue(s). For example, the GOI may be a toxin against common scab—whichnormally affects tuber tissue.

Third, large quantities of the product of expression of a GOI where theGOI is, for example, a desired compound of benefit to humans or animals(e.g. a desirable foodstuff or an enzyme having a beneficialpharmaceutical effect) can be achieved. Furthermore, that product iseasily retrievable.

Fourth, use of the promoter according to the present invention enablesone to express a suitable nucleotide in order to change the organism'smetabolism at a specific site—such as increasing starch levels in tuberor even producing modified starch therein.

A further surprising advantage is that the promoter of the presentinvention, in particular the promoter of the first aspect of the presentinvention, is cold-inducible—i.e. leads to expression in conditions ofabout from 0° C. to 12° C., to about 4° C. Thus this promoter is veryuseful for expressing GOI's in conditions that would be of some benefitin cold conditions—in particular such as expression of the alpha—amylasegene (or active fragment thereof) of EP-B-0470145 (shown as SEQ IDNO:18). More preferably the GOI is a nucleotide sequence that isanti-sense to that alpha-amylase gene (or active fragment thereof), suchas that shown as SEQ ID NO:19.

Highly preferred embodiments of each of the aspects of the presentinvention do not include the native promoter in its natural environment.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site in the Jacob-Monod theory of gene expression.The promoters of the present invention are capable of expressing a GOI.In addition to the nucleotide sequences described above, the promotersof the present invention could additionally include conserved regionssuch as a Pribnow Box or a TATA box. The promoters may even containother sequences to affect (such as to maintain, enhance, decrease) thelevels of expression of the GOI. For example, suitable other sequencesinclude the Sh1-intron or an ADH intron. Other sequences includeinducible elements—such as temperature, chemical, light or stressinducible elements. Also, suitable elements to enhance transcription ortranslation may be present. An example of the latter element is the TMV5′ leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson PlantMol. Biol. 23 [1993] 97). The promoter of the present invention may alsobe called Amy 3 promoter or Amy 351 promoter or alpha-Amy 351 promoteror alpha-Amy 3 promoter.

In addition the present invention also encompasses combinations ofpromoters or elements.

For example, a promoter of the present invention, such as a tuberspecific promoter (see above), may be used in combination with a stemspecific promoter (see above). Other combinations are possible. Forcxample, the promoter of the present invention, such as a stem or tuberspecific promoter, may be used in combination with a root promoterand/or a leaf promoter.

The term “corresponding” in relation to the present invention means thatthe promoter sequence need not necessarily be derived from Solanumtuberosum. For example, the promoter could be prepared synthetically. Itmay even be derived from another source.

The terms “variant”, “homologue” or “fragment” include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence has the ability to act as a promoterin an expression system—such as the transformed cell or transgenicorganism according to the present invention. In particular, the term“homologue” covers homology with respect to structure and/or functionproviding the resultant nucleotide sequence has the ability to act as apromoter. With respect to sequence homology, preferably there is atleast 75%, more preferably at least 85%, more preferably at least 90%homology, more preferably at least 95%, more preferably at least 98%homology.

The term “inactivated” means partial inactivation in the sense that theexpression pattern of the complete promoter of FIG. 8 is modified butwherein the partially inactivated promoter still functions as apromoter. However, as mentioned above, the modified promoter is capableof expressing a GOI in at least one (but not all) specific tissue of thecomplete promoter of FIG. 8. Therefore with this particular aspect ofthe invention, the promoter having an inactivated portion can stillfunction as a promoter (hence it is still called a promoter) but whereinthe promoter is capable of expressing a GOI in one or more, but not all,of the tissues where a GOI is expressed by the complete promoter shownin FIG. 8.

Examples of partial inactivation include altering the folding pattern ofthe promoter sequence, or binding species to parts of the nucleotidesequence, so that a part of the nucleotide sequence is not recognisedby, for example, RNA polymerase. Another, and preferable, way ofpartially inactivating the promoter is to truncate it to form fragmentsthereof. Another way would be to mutate at least a part of the sequenceso that the RNA polymerase can not bind to that part or another part.

Accordingly, for a preferred embodiment of the present invention thereis provided a promoter comprising a nucleotide sequence corresponding tothe 5.5 Kb EcoR1 fragment isolated from Solanum tuberosum, or a variant,homologue or fragment thereof but wherein the promoter is truncated. Theterm “truncated” includes shortened versions of the promoter shown inFIG. 8.

Accordingly, for a preferred embodiment of the present invention thereis also provided a promoter comprising a nucleotide sequencecorresponding to the 5.5. Kb EcoR1 fragment isolated from Solanumtuberosum, or a variant, homologue or fragment thereof, but wherein thepromoter does not contain at least the nucleotide sequence of any of onethe sequences shown as SEQ ID NOS:4-16.

Furthermore, for a preferred embodiment of the present invention thereis also provided a promoter comprising a nucleotide sequencecorresponding to the 5.5. Kb EcoR1 fragment isolated from Solanumtuberosum, or a variant, homologue or fragment thereof, but wherein thepromoter does not contain at least the nucleotide sequence shown as SEQID NOS:1.

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a GOI directly or indirectly attachedto the promoter. An example of an indirect attachment is the provisionof a suitable spacer group such as an intron sequence, such as theSh1-intron or the ADH intron, intermediate the promoter and the GOI. Thesame is true for the term “fused” in relation to the present inventionwhich includes direct or indirect attachment. In each case, it is highlypreferred that the terms do not cover the natural combination of thewild type alpha amylase gene ordinarily associated with the wild typegene promoter and the wild type promoter and when they are both in theirnatural environment.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a plant cell intowhich it has been transferred. Various markers exist which may be usedin, for example, plants—such as mannose. Other examples of markersinclude those that provide for antibiotic resistance—such as resistanceto G418, hygromycin, bleomycin, kanamycin and gentamycin.

The term “GOI” with reference to the present invention means any gene ofinterest. A GOI can be any nucleotide that is either foreign or naturalto the organism (e.g. plant) in question.

Typical examples of a GOI include genes encoding for proteins andenzymes that modify metabolic and catabolic processes. For example, theGOI may be a protein giving added nutritional value to the plant as afood or crop. Typical examples include plant proteins that can inhibitthe formation of anti-nutritive factors and plant proteins that have amore desirable amino acid composition (e.g. a higher lysine content thanthe non-transgenic plant).

The GOI may even code for an enzyme that can be used in food processingsuch as chymosin, thaumatin and alpha-galactosidase. The GOI may evencode for an agent for introducing or increasing pathogen resistance. TheGOI may even be an antisense construct for modifying the expression ofnatural transcripts present in the relevant tissues.

The GOI may even code for a non-natural plant compound that is ofbenefit to animals or humans. For example, the GOI could code for apharmaceutically active protein or enzyme such as any one of thetherapeutic compounds insulin, interferon, human serum albumin, humangrowth factor and blood clotting factors. In this regard, thetransformed cell or organism could prepare acceptable quantities of thedesired compound which would be easily retrievable from, for example,the tubers.

Preferably the GOI is a gene encoding for any one of a protein having ahigh nutritional value, a pest toxin, an antisense transcript such asthat for patatin, ADP-glucose pyrophosphorylase (e.g. see EP-A-455316),alphα-amylase (e.g. see EP-B-0470145), a protease antisense or aglucanase. A preferred GOI is an anti-sense sequence to thealphα-amylase gene described in EP-B-0470145.

The term ‘organism’ in relation to the present invention includes anyorganism that can activate the promoter of the present invention, suchas amylase (e.g. alphα-amylase) producing organisms including plants,algae, fungi and bacteria, as well as cell lines thereof. Preferably theterm means a plant or cell thereof, preferably a dicot, more preferablya potato.

The term ‘transgenic organism’ in relation to the present inventionmeans an organism comprising either an expressable construct accordingto the present invention or a product of such a construct. For examplethe transgenic organism can comprise an exogenous nucleotide sequence(e.g. GOI as herein described) under the control of a promoter accordingto the present invention; or a native nucleotide sequence under thecontrol of a partially inactivated (e.g. truncated) promoter accordingto the present invention.

The terms “cell”, “tissue” and “organ” include cell, tissue and organper se and when within an organism. For one class/type of promotersaccording to the present invention the term means potato tuber cell,tissue or organ and/or potato root cell, tissue or organ and/or potatosprout cell, tissue or organ and/or potato stem cell, tissue or organ.Preferably, the term means means just or at least a potato tuber cell,tissue or organ.

Preferably the expressable construct is incorporated in the genome ofthe organism. The term incorporated preferably covers stableincorporation into the genome.

The term ‘nucleotide’ in relation to the GOI includes genomic DNA, cDNA,synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA.

The term “expression vector” means a construct capable of in vivo or invitro expression.

The term “transformation vector” means a construct capable of beingtransferred from one species to another—such as from an E. Coli plasmidto a plant cell.

Even though the promoters of the present invention are not disclosed inEP-B-0470145 and CA-A-2006454, those two documents do provide someuseful background commentary on the types of techniques that may beemployed to put the present invention into practice.

Some of these background teachings are included in the followingcommentary.

The basic principle in the construction of genetically modified plantsis to insert genetic information in the plant genome so as to obtain astable maintenance of the inserted genetic material. Several techniquesexist for inserting the genetic information, the two main principlesbeing direct introduction of the genetic information and introduction ofthe genetic information by use of a vector system. A review of thegeneral techniques may be found in articles by Potrykus (Annu Rev PlantPhysiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27).

Thus, in one aspect, the present invention relates to a vector systemwhich carries a promoter or construct according to the present inventionand which is capable of introducing the promoter or construct into thegenome of a plant such as a plant of the family Solanaceae, inparticular of the genus Solanum, especially Solanum tuberosum.

The vector system may comprise one vector, but comprises preferably twovectors; in the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells with agiven promoter or construct is based on the use of a Ti plasmid fromAgrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenesAn et al. (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al.(1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S.Ingrams and J. P. Helgeson, 203-208.

Several different Ti and Ri plasmids have been constructed which aresuitable for the construction of the plant or plant cell constructsdescribed above. A non-limiting example of such a Ti plasmid is pGV3850.

The promoter or construct of the present invention should preferably beinserted into the Ti-plasmid between the terminal sequences of the T-DNAor adjacent a T-DNA sequence so as to avoid disruption of the sequencesimmediately surrounding the T-DNA borders, as at least one of theseregions appear to be essential for insertion of modified T-DNA into theplant genome.

As will be understood from the above explanation, the vector system ofthe present invention is preferably one which contains the sequencesnecessary to infect a plant (e.g. the vir region) and at least oneborder part of a T-DNA sequence, the border part being located on thesame vector as the genetic construct. Furthermore, the vector system ispreferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacteriumrhizogenes Ri-plasmid or a derivative thereof, as these plasmids arewell-known and widely employed in the construction of transgenic plants,many vector systems exist which are based on these plasmids orderivatives thereof.

In the construction of a transgenic plant the promoter or construct maybe first constructed in a microorganism in which the vector canreplicate and which is easy to manipulate before insertion into theplant. An example of a useful microorganism is E. coli, but othermicroorganisms having the above properties may be used. When a vector ofa vector system as defined above has been constructed in E. coli, it istransferred, if necessary, into a suitable Agrobacterium strain, e.g.Agrobacterium tumefaciens.

The Ti-plasmid harbouring the promoter or construct of the invention isthus preferably transferred into a suitable Agrobacterium strain, e.g.A. tumefaciens, so as to obtain an Agrobacterium cell harbouring thepromoter or construct of the invention, which DNA is subsequentlytransferred into the plant cell to be modified.

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson, 203-208. See also Potrykus (Annu RevPlant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27).

As reported in CA-A-2006454, a large amount of cloning vectors areavailable which contain a replication system in E. coli and a markerwhich allows a selection of the transformed cells. The vectors containfor example pBR 332, pUC series, M13 mp series, pACYC 184 etc. In such away, the construct or promoter can be introduced into a suitablerestriction position in the vector. The contained plasmid is used forthe transformation in E. coli. The E. coli cells are cultivated in asuitable nutrient medium and then harvested and lysed. The plasmid isthen recovered. As a method of analysis there is generally used asequence analysis, a restriction analysis, electrophoresis and furtherbiochemical-molecular biological methods. After each manipulation, theused DNA sequence can be restricted and connected with the next DNAsequence. Each sequence can be cloned in the same or different plasmid.After each introduction method of the desired promoter or construct inthe plants further DNA sequences may be necessary. If for example forthe transformation, the Ti- or Ri-plasmid of the plant cells is used, atleast the right boundary and often however the right and the leftboundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of theintroduced genes, can be connected. The use of T-DNA for thetransformation of plant cells is being intensively studied and is welldescribed in EP 120 516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley,et al., Crit. Rev. Plant Sci., 4:1-46 and An et al., EMBO J. (1985)4:277-284.

Direct infection of plant tissues by Agrobacterium is another simpletechnique which may be employed. Typically, a plant to be infected iswounded, e.g. by cutting the plant with a razor or puncturing the plantwith a needle or rubbing the plant with an abrasive. The wound is theninoculated with the Agrobacterium, e.g. in a solution. Alternatively,the infection of a plant may be done on a certain part or tissue of theplant, i.e. on a part of a leaf, a root, a stem or another part of theplant. The inoculated plant or plant part is then grown on a suitableculture medium and allowed to develop into mature plants.

When plant cells are constructed, these cells may be grown andmaintained in accordance with well-known tissue culturing methods suchas by culturing the cells in a suitable culture medium supplied with thenecessary growth factors such as amino acids, plant hormones, vitamins,etc. Regeneration of the transformed cells into genetically modifiedplants may be accomplished using known methods for the regeneration ofplants from cell or tissue cultures, for example by selectingtransformed shoots using an antibiotic and by subculturing the shoots ona medium containing the appropriate nutrients, plant hormones, etc.

In summation therefore the present invention therefore relates to apromoter and, also to a construct comprising the same. In particular thepresent invention relates to the use of a promoter for the expression ofa GOI in an cell/tissue/organism such as one or more specific tissues ofa plant, in particular a dicot plant such as a potato.

More in particular, in a preferred embodiment, the present inventionrelates to a partially inactivated (such as truncated) type 3 α-amylasepromoter.

The present invention also relates to the application of one class ofpartially inactivated gene promoters to express a GOI specifically inthe tuber tissue of a dicot—especially a potato plant.

The following sample has been deposited in accordance with the BudapestTreaty at the recognised depositary The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive,Aberdeen, Scotland, AB2 1RY, United Kingdom, on Aug. 26, 1994:

DH5alpha-gPAmy 351 (Deposit No. NCIMB 40682).

This sample is an E. Coli bacterial stock containing the plasmidpBluescript (see FIG. 7 for a general map thereof) containing an EcoR15.5 genomic DNA fragment isolated from potato (Solanum tuberosum). TheEcoR1 5.5 fragment contains the promoter region and part of the 5′untranslated sequence of the structural gene of a potato alphα-amylasegene. The plasmid was formed by inserting the EcoR1 5.5 kb potatofragment into the polylinker of the vector pBS (Short et al [1988] Nuc.Acid. Res. 16:7583-7600). The promoter may be isolated from the plasmidby enzyme digestion with EcoR1 and then extracted by typical separationtechniques (e.g. gels).

The following sample has been deposited in accordance with the BudapestTreaty at the recognised depositary The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive,Aberdeen, Scotland, AB2 1RY, United Kingdom, on Oct. 20, 1994:

DH5alpha-pJK4 (Deposit No. NCIMB 40691).

This sample is an E. Coli bacterial (DH5alpha-) stock containing plasmidpJK4 (described later).

The present invention will now be described only by way of examples inwhich reference is made to the Figures.

In more detail, FIG. 1 is a restriction enzyme map of genomic clonegPAmy 351 isolated from the potato variety Saturna, in which the arrowindicates the position of the promoter, the closed bar indicates theposition of coding sequences, H=HindIII, E=EcoRI, S=SalI, ATG=initiationcodon of the alphα-amylase coding sequence and A star marks the positionof the 5.5 kb EcoRI fragment.

FIG. 2 is a sequence map of the alpha-Amy 3 promoter in which the arrowsshow the extent of the sequence reactions, the position of the HEfragment is shown in B together with the 5′ sequenced part of thepromoter deletion series, the names of the individual fragments (seealso FIG. 4) are given above the arrows, ATG=initiation codon of thealphα-amylase coding sequence, and the deletion fragments chosen forfunctional analysis are indicated by asterisks.

FIG. 3 is a nucleotide sequence of part of the alpha-Amy 3 promoter inwhich the restriction sites are bold faced, TATA, CCAAT and ATG sitesare underlined, the position of the proposed CAP site and theuntranslated leader sequence are indicated, and the 166 bp nucleotidesequence sandwiched between the two highlighting lines (i.e. fromnucleotide position −857 to nucleotide position −691) is represented asSEQ. I.D. No. 1 (see later). This 166 bp nucleotide sequence may bereferred to as the “delta” fragment or sequence.

FIGS. 4 and 5 represent two deletion series of the alpha-Amy 3 promoterwith those of FIG. 5 being used for functional analysis; FIG. 6 shows aseries of primer sequences for use with the present invention whereinUni=T7 primer and Rev=T3 primer.

In further detail, the nucleotide sequence of FIG. 3 is part of thepromoter sequence of FIG. 1 (discussed below) and part of thealphα-amylase structural gene, which in turn is part of the sequence ofFIG. 8. Part of this nucleotide sequence forms part of the sequencesshown in the attached sequence listings. The nucleotide sequence of FIG.3 is repeated as SEQ ID NOS:17.

FIG. 8 is a pictorial representation of plasmid gPAmy351. Thehighlighted portion is a EcoR1-SalI fragment isolated from potato(Solanum tuberosum)—which is the same as the fragment shown in FIG. 1.The EcoR1-SalI fragment contains the EcoRI 5.5 kb fragment (calledsubclone Eco 5.5)—which is indicated by the line shown at the bottom ofthe drawing. The EcoRI 5.5 kb fragment contains the promoter region andpart of the 5′ untranslated sequence of the structural gene of a potatoα-amylase. The following restriction enzyme sites are shown in FIG. 8:E: EcoRI, Ha: HaeIII, Sp: Sspl, H: HindIII, P: Pvul, S: SalI. Inaddition putative CAAT and TATA boxes and the ATG initiation site areshown. Introns are shown as open bars and exons as closed bars.

The EcoRI 5.5 fragment is cloned into a pBluescript M13-plasmid (shownin FIG. 7) or a pBSK-plasmid (also shown in FIG. 7).

For convenience, Chart 1 correlates the sequence references shown in theattached Figures with the sequences shown in the attached SequenceListings.

CHART 1 SEQUENCE I.D. No. FIGURE No./FIGURE REFERENCE 1 4/Delta 2 4/EH 34/8.5-E 4 4/9.5-7 5 4/8-17 6 4/7-1 7 4/6-15 8 4/6-13 9 4/6.5-4 10 4/5-24 11  4/4-1 12  4/4-2 13  4/1-8 14  4/1-6 15  EH8 16  −/− 17  3 25/HE 3 5/HFP8 7 5/HFP6 11  5/HFP4 15  5/EH8

In the following examples, the following materials and methods were usedand followed, respectively.

MATERIALS AND METHODS

Plant Material

Root tissue were harvested from flowering potato (Solanum tuberosum, cv.Saturna) plants. The roots were sliced directly into liquid nitrogen and10-15 g portions were stored at −80° C. until use.

Bacterial Strains

DH5α™ (BRL): F⁻, enA1, hsdR17(r_(k)-, m_(k)+), supE44, thi-1, γ⁻, recA1,gyrA96, relA1, (argF-lacZYa)U169, σ80dlacZ ΔM15

JM109(1): recA1, endA1, gyrA96, thi, usdR17, supE44, relA1, γ⁻Δ(lac⁻proAB), [F′, traD36, proAB, LacI^(q)Z ΔMI5]

PLK17 (Stratagene): hsdR-M+, mcrA-, B-, lac-, supE, gal

LE392 (2,3): supE44, supF58, hsdR514, galK2, galT22, metB1, trpR55,lac41

LBA4404: contains the disarmed pTiAch5 plasmid pAL4404 in thestreptomycin resistant derivative of the Agrobacterium tumefaciensstrain Ach5 (4).

Phages and plasmids λ EMBL3: see referenee (5) pBR327: see referenee (6)pBS+, pBS−: see reference (7) pBSK+, pBSK−: see referenee (7) pBI101,pBI121: see reference (8, 9)

Media and Plates

L-Broth (LB) medium:

Per litre: 5 g of yeast extract, 5 g of NZ-amide, 5 g of NaCl, 5 g ofbacto-peptone. Autoclave.

LB-plates:

LB medium plus 15 g Bacto agar per litre. Autoclave. Pour into plasticpetri dishes (25 ml/dish).

Amp-plates:

As LB-plates plus 35 mg ampicillin per litre after autoclaving.

AXI-plates:

As LB-plates plus 35 mg ampicillin, 120 mg IPTG(isopropylthiogalactoside), 40 mg

Xgal (dissolved in dimethylformamide) per litre after autoclaving.

Xgal: 5-bromo-4chloro-3indolyle-β-D-galactoside.

Kan-plates:

As LB-plates plus 50 mg kanamycin per litre after autoclaving.

YMB medium:

Per litre:0.66 g K2HPO4-3H20. 0.2 g MgSO4. 0.1 g NaCl. 10.0 g Mannitol.0.4 g

Yeast extract. Adjust pH to 7.0. Autoclave.

Liquid MBa medium:

Per litre:4.4 g MS salts (Murashige and Skoog basal salt (10),Sigma). 20g sucrose. pH is adjusted to 5.7 with NaOH.

Solid MBa medium:

As liquid MBa medium plus 0.8% Difco agar.

MBa co:

As solid MBa medium plus 0.5 mg t-Zeatin (trans-isomer, Sigma) and 2.0mg 2,4 D (2,4-dichlorophenoxacetic acid, Sigma) per litre.

Solid MBb medium:

As solid MBa medium but instead of 20 g sucrose 30 g sucrose is addedper litre.

Water:

The water used in Materials and Methods was always distilled andautoclaved before use.

Isolation of High MW Genomic Potato DNA

In order to gain high molecular weight genomic DNA a procedureessentially as described by Fischer and Goldberg (11), was followed.This include first isolation of nuclei followed by preparation of thenuclear DNA.

10-15 g Saturna root tissue were ground to a fine powder in liquidnitrogen and homogenized in 100 ml H buffer (1×H buffer(11): 100 ml10×HB, 250 ml 2M sucrose, 10 ml 100 mM PMSF, 1 ml β-mercaptoethanol, 5ml Triton X-100, 634 ml H₂O. Adjust to pH 9.5. Add β-mercaptoethenoljust before use. 10×HB: 40 mM spermidine, 10 mM spermine, 0.1 mMNa-EDTA, 0.1 mM Tris, 0.8 mM KCl, adjusted to pH 9.4-9.5 with 10N NaOH.PMSF: phenylmethylsulfonyl fluoride dissolved in ethanol). Theresuspended plant material was filtered through a 70 μm nylon filter(Nitex filter, prewetted in 1×H buffer). The resulting filtrate waspoured into two centrifuge bottles (Sorvall GSA) and the nuclei werepelleted at 4000 r.p.m for 20 min at 4° C. The supernatant was discardedand the pellets were gently resuspended by adding 20 ml 1×H buffer pertube and then swirling the tubes carefully. The nuclei were pelletedagain at 4000 r.p.m. for 20 min at 4° C., the supernatant removed andthe pellets resuspended gently in 10 ml 1×H buffer. The supernatant werepooled and 20 ml cold lysis buffer (lysis buffer: 2% Sarcosyl, 0.1MTris, 0.04M Na₂-EDTA) was added dropwise while the solution was stirredgently. Immediately after the last drop of lysis buffer was added, 0.972g CsCl/ml solution was stirred gently into the solution (the solutionshould now be at room temperature). The resulting solution wascentrifuged for 45 min at 10 krpm, 4° C. The supernatants were carefullyremoved using a pasteur pipet avoiding any protein debris floating onthe top or disturbing the pellets. The volume of the supernatants weredetermined and 0.2 mg ethidium bromide/ml was added. The DNA solutionwas gently poured into quickseal polyallomer tubes, which were thensealed. The tubes were centrifuged in a Beckman VTI 65 rotor at 18° C.and 40 k r.p.m. for 38 h. The genomic band was removed under UV-lightwith a 15-18 gauge needle attached to a 5-ml syringe and poured gentlyinto a 5 ml polyallomer tube.

The tube(s) was then filled with a 1.57 g/ml CsCl solution in 5 mMTris-Hcl(pH 9.5), 20 mM Na₂-EDTA. 75 μl ethidium bromide (5 mg/ml) wasadded/tube. The tubes were centrifuged in the VTI 65 rotor at 18° C. and46 k r.p.m. for 17 h. The genomic band was removed under long-wave UVlight and the ethidium bromide was extracted with CsCl-saturatedisopropanol (7 to 8 times).

The CsCl was removed from the DNA by dialysis in TE-buffer (1×TE: 10 mMTris-HCl, 1 mM Na₂-EDTA pH 8.0) at 4° C. for 18 h with three changes.The high MW genomic potato DNA was not further precipitated and was keptat 4° C.

Construction of a Potato Genomic Library

High MW genomic potato DNA was prepared from cv Saturna roots asdescribed above. The quality of the DNA was tested by restriction enzymedigestion and gel electrophoresis.

The genomic DNA was partially digested with Sau3A and the createdfragments (9-23 kb) were inserted into the BamHI site of the λ EMBL3vector(4). Approximately 1.1×10⁶ independent isolates were plated andamplified to form a permanent library (12). Plaque hybridization wasused to screen the library for α-amylase genes.

Screening of the Library

Screening of the potato genomic library was carried out essentially asdescribed by references 13 and 14. The pfu/ml (pfu:plaque forming unit)of the amplified genomic library was determined in duplicate prior tothe screening. Infection competent cells (PKL17 or LE392) were preparedby inoculating the cells in 30 ml fresh L-Broth containing 0.2% sucroseand 10 mM CaCl₂. The cells were cultivated for 4-5 h at 37° C. before0.1 vol of cold CaCl₂ was added and kept on ice until use. 100 μl phagesdiluted in phagebuffer to give an appropriate number of pfu(1×phagebuffer: 10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 20 mM NaCl) weremixed with 25-100 μl freshly made cells (dependent on the actual numberof cells) and incubated at 37° C. for 15-20 min. The suspension wasmixed with 3 ml warm (42° C.) 0.8-1% top agar containing 10 mM MgCl₂ andplated out on dry LB plates.

LB plates of 2×22 cm (dried for 3-4 h at 37° C.) were used for screeningof the genomic library. Each plate contained approximately 2×10⁵plaques, which were mixed with 1 ml of infection competent cells(prepared as above) and incubated for 20 min at 37° C. This mixture wasthen added to a 25 ml of warm (42-45° C.) 0.3% top agarose with 10 mMMgCl₂ and the solution was poured onto a fresh dry LB plate. The largeLB plates were incubated (not upside down) overnight at 37° C. Phagesfrom the plaques were transferred to Hybond N filters (Amersham) induplicates. The plates were placed at 4° C. for 1 to 2 h to prevent theagarose layer from sticking to the filters.

The plates were placed on ice, just before use and they remained on theice when working with the filters. Two Hybond N filters and a plate weremarked for orientation of the filters . The first filter was laid on theplaques for 45 sec; then floated on denaturation buffer (0.5 M NaOH, 1.5M NaCl) for 7 min, with the phages facing up, then floated onneutralization buffer (0.5 M Tris-HCl (pH 7.4), 3 M NaCl) for 2 times 3min and finally washed in 2×SSC (1×SSC: 0.15 M NaCl, 0.015 Na-citrate).The filter was air dried and the phage DNA was fixed to the membrane byUV crosslinking. The second filter was laid on the same plate, after thefirst, for 120 sec and then treated as the first. These filters wereused in plaque hybridization following the Hybond N membrane protocolaccording to suppliers (Amersham) instructions. X-ray film from both thefirst and second Hybond N membrane was orientated so that the signalsfrom both filters fitted each other.

The positive plaques were cut with a scalpel (1×1 cm blocks) andsubmerged in 1 ml phagebuffer. The phage containing tubes were storedairtight (parafilm) at 4° C. after 2-3 drops of chloroform has beenadded. The plaque containing plates (22×22 cm) were stored by placing apiece of soaked (chloroform) filter paper in the lid. The plates werealso stored airtight at 4° C. with the plaques facing up. Furtherpurification of the positive plaques were done by plating dilutions ofthe stock tube (containing the 1×1 cm block) with freshly prepared cellsand plate them on round LB plates with 1% warm (42° C.) top-agar and 10mM MgCl₂.

New filter prints were made with Hybond N following the procedureoutlined above with the 22×22 cm plates. Plaques which gave positivesignal were isolated by sticking the tip of a pasteur pipette though theplate and transfer it to 500 μl phage buffer.

A new series of dilutions were made, plated and the respective filtershybridized until the positive plaques were purified. The phages werestored airtight, at 4° C. either in the 500 μl phagebuffer with 1 dropof chloroform, or as phages isolated from a plate lysate. The platelysate stock was made as described by (14).

Isolation of Recombinant γ DNA

Large-scale preparations followed the method described in (14), whichinclude banding the recombinant phage DNA on a CsCl gradient. Twoversions (A,B) of a small-scale preparation were used as follows:

A) LE392 cells were inoculated in LB with 0.2% maltose and 10 mM MgCl₂and grown O/N at 37° C. The cells were pelleted by centrifugation for 10min, at 4° C. in a Sorvall centrifuge, and resuspended gently in 1volume of cold 10 mM MgSO₄. The cells were stored at 4° C. until use.Five single plaques from a plate were transferred to 500 μl phagebufferand allowed to stand for 2-2½ h at 4° C. After votex of the tube 100 μlof the liberated phages were mixed with 200 μl freshly prepared LE392cells. Alternatively 50-100 μl liberated phages from aplate lysate weremixed with the cells. Phages and cells were incubated for 20 min at 37°C. and then added to a prewarmed (37° C.) 25 ml LB with 20 mM MgSO₄ and30 mM Tris-HCl pH 7.5 and incubated, shaking O/N at 37° C. A further 10ml prewarmed LB with 20 mM MgCl₂ and 30 mM Tris-HCl pH 7.5 was added andthe mix incubated for 1-2 h shaking at 37° C. After clear lysis(eventuel a few drops of chloroform was added to help) and the solutionwas centrifuged at 8000 r.p.m. for 10 min at 4° C. The supernatant wastransferred to a new tube and centrifuged again if necessary to removecell debris.

The recombinant γ DNA was then purified using a Qiagen column followingthe suppliers instructions (15).

B) The procedure was as under A) until after the first centrifugation ofthe O/N culture. The supernatant was transferred to a new tube and DNasewas added corresponding to 1 μg/ml. The solution was incubated 30 min at37° C. and then 1 volume of cold 20% PEG, 2 M NaCl mixed in phagebufferwas added and the mixture was incubated 1 h on ice. The phages werepelleted by centrifugation for 20 min, 4° C. at 10 krpm. The PEG pelletwas resuspended in 400 μl phagebuffer and transferred to an eppendorftube. 1 μl of RNasc (10 mg/ml) is added and the tube incubated for 30min at 37° C. Then 8 μl 0.25 M Na₂-EDTA, pH 8.0 and 4 μl 10% SDS wereadded, the tube was incubated a further 15 min at 68° C. The mixture wasallowed to gain room temperature and then an equal phenol saturated withTE-buffer (1×TE: 10 mM Tris pH 7.5, 1 mM Na₂-EDTA) was used to extractthe DNA. A equal mixture of saturated phenol-chloroform was used toextract the upper aqueous phase and a final chloroform extraction wasdone. The upper phase was transferred to a new tube and the solution wasmade 0.3 M Na-acetate and 2-3 vol cold ethanol was added. Theprecipitation of the DNA was accomplished by storing at O/N at −20° C.,centrifuging for 5 min and resuspend the pellet in 50-100 μl TE-buffer.The amount and quality of the recombinant phage DNA was tested byrestrictions enzyme digest and agarose (0.8-1%) gel electrophoresis(16).

Preparation of Plasmid DNA

The plasmid preparation was as described in EP-B-0470145. In particular,small scale preparation of plasmid DNA was performed as follows.Bacterial strains harbouring the plasmids were grown overnight in 2 mlL-Broth (LB) medium with ampicillin added (35 μg/ml).

The operations were performed in 1.5 ml Eppendorf tubes andcentrifugation was carried out in an Eppendorf centrifuge at 4° C. Thecells from the overnight culture were harvested by centrifugation for 2min., washed with 1 ml 10 mM Tris-HCl (pH 8.5), 50 mM EDTA andcentrifuged for 2 min. The pellet was suspended in 150 μl of 15%sucrose, 50 mM Tris-HCl (pH 8.5), 50 mM EDTA by vortexing. 50 μl of 4mg/ml lysozyme was added and the mixture was incubated for 30 min. atroom temperature and 30 min. on ice. 400 μl ice cold H20 was added andthe mixture was kept on ice for 5 min, incubated at 70-72° C. for 15min. and centrifuged for 15 min. To the supernatant was added 75 μl 5.0M Na-perchlorate and 200 μl isopropanol (the isopropanol was stored atroom temperature), and the mixture was centrifuged for 15 min. at 4° C.The pellet was suspended in 300 μl 0.3 M Na-acetate and 2-3 vol. coldethanol was added. Precipitation was accomplished by storing at either 5min. at −80° C. or O/N at −20° C., centrifuging for 5 min., drying byvacuum for 2 min. and redissolving the pellet in 20 μl H₂O. The yieldwas 5-10 μg plasmid DNA.

Large scale preparation of plasmid DNA was accomplished by simplyscaling up the small scale preparation ten times. Working in 15 ml corextubes, all the ingredients were scaled up ten times. The centrifugationwas carried out in a Sorvall cooling centrifuge at 4° C. Only changesfrom the above will be mentioned in the following. After incubation at70-72° C., the centrifugation was for 30 min. at 17,000 rpm. Afteradding isopropanol and after adding cold ethanol, the centrifugation wasfor 15 min. at 17,000 rpm. The final plasmid DNA pellet was suspended inH₂O and transferred to an Eppendorf tube and then given a short spin toremove debris. The supernatant was adjusted to 0.3 M Na-acetate and 2-3vol. cold ethanol were added. The pellet was resuspended in 40 μl H₂O.The yield was usually 20-28 μg plasmid DNA.

To obtain very pure plasmid DNA, 200-300 μg of isolated plasmid DNA fromthe upscaled method were banded on a CsCl gradient. Solid CsCl was mixedwith H₂O (1:1 w/v) and 0.2 mg/ml ethidium bromide was added. Thesolution was poured into a quick-seal polyallomer tube and the plasmidDNA, mixed with solid CsCl (1:1 w/v). The tube was filled, sealed andcentrifuged in a Beckman VTI 65 rotor at 15° C., 48,000 rpm for 16-18hours. The centrifuge was stopped by without using the brake. The bandedplasmid DNA was withdrawn from the tubes using a syringe and theethidium bromide was extracted with CsCl-saturated isopropanol 7-8times. The CsCl was removed by dialysis in 10 mM Tris-HCl (pH 8.0), 1 mMEDTA for 48 hours with three changes of buffer. The DNA was precipitatedby adjusting to 0.3 M Na-acetate and adding 2-3 vol. cold ethanol.

The small scale plasmid preparation from E. coli was usually followed bya LiCl precipitation to remove RNA from the DNA solution. The smallscale prepared plasmid DNA was dissolved in 100 μl destilled water. 1vol of SM LiCl was added and the mixture incubated at −20° C. for 30 minfollowed by centrifugation at 12,000 rpm. for 15 min, 4° C. Thesupernatant was transferred to a new eppendorf tube and 2 vol TE bufferor water was added. Precipitation with 2.5 vol of 96% ethanol wasaccomplished by storing either 10 min. at −80° C., or O/N at −20° C. TheDNA was precipitated by centrifuging for 15 min. 12,000 rpm ,at 4° C.,drying by vacuum for 2 min and redissolving in 18 μl of TE or water.

Restriction Enzyme Digestion

The protocol followed was that outlined in EP-B-0470145. In particular,all restriction endonucleases were from Biolabs, Amersham or BoehringerMannheim and were used according to the supplier's instructions. 1 unitof enzyme was used to 1 μg of DNA and incubation was for 2 hours.

The buffer was changed in double digestions, by changing the volume orby adding the necessary ingredient according to the enzyme instructions.

Labelling of DNA

A random primed DNA labelling kit (Boehringer Mannheim) was usedaccording to the suppliers instructions. Briefly, 2 μl DNA fragment(25-50 ng) is mixed with 8 μl H₂O and incubated at 95° C. for 10 min todenature the DNA. Spin shortly and place on ice. Then add 1 μl dGTP,DATP and dTTP of each, 2 μl reactionsmix and 5 μl (approx. 50 μCidCTP³²). 1 μl Klenow enzyme starts the reaction and the tube isincubated at 37° C. for 30 min. Then place on ice. The labelled DNAfragment was purified using an ELUTIP column (Schleicher & Schuell). Thecolumn was prepared by prerunning (gravity) it with 3 ml high saltbuffer (1.0 M NaCl, 20 mM Tris-HCl (pH 7,5), 1.0 mM EDTA), followed by 5ml low salt buffer (0.2 M NaCl, 20 mM Tris-HCl (pH 7,5), 1.0 mM EDTA).250 μl low salt was added to the labelling tube and the entire solutionwas laid on the prepared column. Then the column was washed with 2×400μl low salt followed by 3×200 μl high salt. The eluted radioactive probewas then heat denatured and used in hybridization.

Southern Transfer and Hybridization

The DNA fragments to be transferred were fractionated on non-denaturingagarose gels (14) and transferred to either Hybond™ N or Hybond™ N+,positively charged nylon membrane (Amersham Life Science) by Southernblotting (17,18). Hybridization to the Hybond™ N nylon membranesfollowed the supplier instructions (18).

Preparation of Vectors

The preparation of vectors was as described in EP-B-0470145 as follows:Vectors (pBS−/+ or pBSK−/+) were digested with one or two restrictionenzymes, extracted twice with saturated phenol (the phenol was firstmixed with 0.1 M Tris-HCl, then mixed twice with TE-buffer (10 mMTris-HCl, pH 8, 1 mM Na₂-EDTA)) and once with chloroform andprecipitated with 0.3 M Na-acetate and 2.5 vol cold ethanol. The pelletwas rinsed in 70% cold ethanol and dissolved in H₂O, giving aconcentration of 25-50 ng/μl. The vectors were tested for backgroundbefore use (self-ligation with and without T4-DNA-ligase). If necessarythe vector was treated with Alkaline phosphatase (Boehringer Mannheim)as described by the supplier. After such a treatment the resultingpellet was resuspended in H₂O to give a final concentration of 10 ng/μl.

Ligation

The phage DNA or plasmid comprising a fragment to be subcloned wasdigested with one or more restriction enzymes and run in either a 5%acrylamide gel or an appropriate agarose gel. The fragment to besubcloned was isolated from the gel either by electroelution asdescribed in (14) or using a GENECLEAN II Kit (BIO 101 Inc., La Jolla,Calif.) following the suppliers instructions.

Various ratios of fragment to vector were used (from 2:1 to 5:1, basedon the number of molecules) in the ligation reaction. 1 μl (10-100 ng)of a solution containing the vector was combined with the fragment, 1 μlof T4-ligation buffer (10×(20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 0.6 mMATP, 10 mM dithiotheitol)) and 1 μl of T4-DNA ligase (BoehringerMannheim) were added to a mixture of fragment and vector to a totalvolume of 10 μl. The reaction was incubated at 15° C. O/N if the ligatedDNA fragments had sticky ends.

If the DNA had blunt ends, the incubation was at room temperature for 1hour. The ligation mixture was stored at −20° C. if not usedimmediately, usually 5 μl of the ligation mix was used fortransformation.

DNA fragments treated with a DNA blunting kit (see “subcloning andsequencing”) were ligated following the DNA blunting kit's protocol(Amersham).

Preparation of Competent E. coli Cells and Transformation

This was done according to the protocols laid down in EP-B-0470145 asfollows:

JM109 cells (or DH5α) were inoculated in 4 ml L-Broth made to 10 mMMgSO₄ and 10 mM MgCl₂. The cells were grown O/N at 37° C. 1 ml of theO/N culture was added to 40 ml prewarmed (37° C.) LB medium (with 10 mMMgSO₄ and 10 mM MgCl₂). The culture was shaken at 250-275 rpm for 1 to 2h until the OD₄₅₀ reached 0.8-0.9. The cells were harvested bycentrifugation at 5000 rpm for 10 min at 4° C. The pellet was gentlyresuspended in 30 ml of cold 0.1 M CaCl₂, another centrifugationpelleted the cells again and they were then resuspended in 15 ml of cold0.1 M CaCl₂. The suspension was placed on ice for 20 min followed by acentrifugation as before. Finally, the cells were gently resuspended in3 ml of cold 0.1 M CaCl₂ and placed on ice for at least 1 h before theywere ready to use for transformation (19).

5 μl of ligation mix was combined with 95 μl of cold sterile TCM (10 mMTris-HCl, pH 7.5, 10 mM CaCl₂, 10 mM MgCl₂, and 0.2 ml of the competentcells. The mixture was allowed to stand for at least 40 min on ice, then5 min at 37° C. (or 2 min at 42° C.). The solution was transferred to0.8 ml of L-Broth, 10 mM MgSO4, 10 mM MgCl₂, and incubated for 45 min at37° C. and then plated out on 5 AXI or other plates (as e.g. Amp-plates)at 0.2 ml/plate.

The plates were allowed to stand 10 min before being inverted andincubated O/N at 37° C. They were stored in plastic bags upside down at4° C.

ExoIII/Mung Bean Nuclease Deletions

A deletion series of a subcloned larger genomic fragment was performedusing a ExoIII/Mung Bean Deletion Kit (Stratagene). The subcloneselected for the deletion series was purified by banding twice on a CsClgradient (see “Preparation of plasmid DNA”) to obtain high amounts ofsupercolied plasmid DNA. Generation of the deletions was performed usingthe ExoIII/Mung Bean Deletion kit following the suppliers instructions.The temperature during the ExoIII treatment was 23° C. since at thattemperature approximately 125 bp should be removed per min.

Purification of Primers Following Synthesis on a DNA Synthesizer

The primer was synthesized on a polystyrene support column (AppliedBiosystems, 393 DNAJ/RNA Synthesizer) and was eluted from the columnwith NH₄OH. The column was broken open and 1.5 ml NH₄OH was added to thepolystyrene material in a small glass tube. The mixture was incubated at85° C. for 1 hour followed by 5 min on ice. The supernatant containingthe single stranded DNA was transferred to eppendorf tubes, and theNH₄OH was evaporated in a vacuum centrifuge for at least 3 h. Pellet wasresuspended in 200 μl destilled water and precipitated with 550 μlethanol and 20 μl sodium acetate. The pellet was resuspended in 200 μlwater and precipitation with ethanol and sodium acetate was repeated.Finally the pellet was resuspended in 100-200 μl destilled water and theOD₂₆₀ was measured by a Gene Quant RNA/DNA calculator (Pharmacia) ofsingle stranded DNA is calculated. An OD₂₆₀ of 1 corresponds approx. to33 μg/ml single stranded DNA.

Subcloning and Sequencing

Purified γ DNA was digested with appropriate restrictions enzymes andthe generated fragments were isolated from agarose gels using aGeneClean Kit (BIO 101 Inc., La Jolla, Calif.) according to thesuppliers instructions.

Genomic DNA fragments (or fragments obtained from plasmids) were ligatedinto the polylinker region of the BlueScribe vector pBS−/+ (or pBSK−/+,Stratagene). After transforming an E. coli strain with the ligatedplasmid the recombinant subclones could be selected by plating on AXIplates (they will be white and the nonrecombinant clones will be bluewhen the vector is a pBlueScript plasmid,(107)).

Plasmid DNA from putative subclones were digested with appropriaterestriction enzymes, subjected to gelelctrophoresis and after Southernblotting, hybridized with an appropriate labelled DNA probe, to verifythe origin of the inserted fragment.

The generated pBS genomic DNA subclones were then sequenced according tothe plasmid preparation protocol outlined in EP-B-0470145. In thisregard, the plasmid (double stranded template) to be sequenced waspurified by the plasmid small scale preparation method. The DNA wasdenatured in 0.2 M NaOH (5 min at room temperature) the mixture wasneutralised by adding 0.4 vol of 5 M ammonium acetate (pH 7.5) and thenprecipitated with 4 vol. of cold ethanol (5 min at −80° C.). The pelletwas rinsed with 70° C. cold ethanol and resuspended in 10 μl H₂O.

For subcloning of DNA fragments generated by using an ExoIII/Mung BeanNuclease kit,the fragments were either blunted first or digested with arestriction enzyme, following by blunting.

The blunting of the DNA with an unknown end structure (after theExoIII/Mung Bean treatment) or with cohesive ends was accomplished byusing a DNA blunting kit (Amersham) following the suppliersinstructions. The generated ligated (see “Ligation”) deletions plasmidswere transformed into DH5α competent cells and white colonies, selectedon AXI plates, were analysed for their insert by restriction enzymedigestion and further, by sequencing.

Sequencing was accomplished with a Sequenase™ DNA Sequencing Kit fromUnited States Biochemical Corp., following the sequencing Protocolenclosed in the kit (Sequenase™ :Step by Step Protocols for DNAsequencing with Sequenase, 3rd Edition, United States BiochemicalCorporation PO Box 22400 Cleveland Ohio 44122). The followingmodifications were however made to the suggested protocol. Instead ofadding DTT, Labelling mix and ³⁵SdATP to the annealed DNA mix, 4 ml of³⁵Sequetide (DuPont) was added.

In addition to T3 and T7 primers (Stratagene) a whole range of otherprimers generated on a DNA synthesizer (Applied Biosystems, 392 DNA/RNASynthesizer) were used. 0.5 pmol of primer was used to sequence 1 pmolof plasmid.

The primer sequences are shown in the FIG. 6.

The sequencing reaction were subjected to electrophoresis on 6% or 8%denaturing polyacrylamide gels for 1 to 4 hours at 40 W, then dried by agel drier and autoradiographed for 3-48 hours at room temperature.

The denaturing sequencing gels were made from pre-mixed polyacrylamidesolutions, Gel-Mix 6 and Gel-Mix 8 (GIBCO BRL, Life technologies, Inc)according to the manufacturers instructions.

Preparation of Competent Agrobacterium Cells and Transformation

The LBA 4404 strain was kept at YMB plates (pH 7.0) containing 100 mg/mlof rifampicin (Sigma) and 500 mg/ml of streptomycin (Sigma). 2.5 ml ofLB medium (pH 7.4) was inoculated with the bacteria. The suspension wasleft growing for 24 hours at 28° C. in an incubation shaker at 300-340rpm. The suspension was then diluted 1:9 with LB and incubated foranother 2-3 hours at 28° C. and 300-340 rpm.

When OD was 0.5-1, 25 ml aliquots of the cells were harvested in 50 mltubes in a cooling centrifuge at 10.000 rpm, 5 min, 4° C. The tubes wereplaced on ice and the pellet resuspended in 0.5 ml of 20 mM CaCl₂. 0.1ml aliquots of the resuspended cells were quickly frozen in 1 mlcryotubes in liquid nitrogen and stored at −80° C.

Transformation was accomplished using the freeze-thaw method (20) asfollows:

A 0.1 ml aliquot of CaCl₂ competent LBA 4404 cells was thawed on ice andadded 1 μg of plasmid DNA. The mixture was incubated at 37° C. for 5min. and added 1 ml LB (pH 7.4). Incubation at room temperature withshaking (100 rpm.) for 4 hours was followed by a quick spin at 10.000rpm, 4° C. for 30 sec. The pellet was resuspended in 100 μl LB andplated on a YMB plates containing 50 mg/l of kanamycin (Sigma).

The plates were incubated for 48 hours at 28° C. or until the colonieshad a suitable size.

This was the first round of selection. Only bacteria transformed with aplasmid containing the NPT II gene conferring kanamycin resistance isable to survive on the kanamycin plate.

For the second round of selection six of the obtained colonies weretransferred to a YMB plate containing 100 mg/l of rifampicin, 500 mg/lof streptomycin and 50 mg/l of kanamycin. LBA 4404 is resistant torifampicin and streptomycin and the plasmid confers resistance tokanamycin. The plates were incubated at 28° C. until the coloniesreached a suitable size (approx. 4-5 days).

The colonies were tested for their plasmid content. Plasmid preparationsof the colonies were generated and the DNA was digested with appropriaterestriction enzymes and run on a 1% agarose gel to ensure that theplasmid and the inserted fragment had the right size. The digested DNAwas blotted onto a Hybond N+ membrane and hybridised with an appropriateradioactively labelled probe (a fragment of the plasmid DNA or insert).

Storage of the transformed LBA 4404 was at −80° C. 2 ml LB mediumcontaining 100 mg/l of rifampicin, 500 mg/l of streptomycin and 50 mg/lof kanamycin was inoculated with bacteria and incubated at 28° C. for 48hours with shaking (300-340 rpm). The suspension was diluted 1:1 withsterile 35% glycerol and aliquoted into cryotubes, 800 μl per tube andstored at −80° C.

Transformation of Potato

A culture of the transformed LBA 4404 bacteria were made by inoculating2 ml of YMB (pH 7.0) with the bacteria and incubating at 28° C. for 24hours. The suspension was diluted 1:10 and incubated for another 18hours. The bacteria was centrifuged at 10.000 rph, 4° C. for 10 min. andthe pellet rinsed twice with 2.5 ml of 2 mM magnesium sulfate, beforeresuspension in liquid MBa to an OD660 nm of 0.5.

The potato plant material used for transformation was maintained invitro at MBa medium added 2 μM STS (21,22). By multiplication top shootsas well as nodes were applied, if the leaves were big they were removed.5 shoots per container with 80 ml medium was left growing at 25° C. and30-35 days after subcultivation the nodes could be used fortransformation.

The stems of micropropagated plants were cut just above and beneath thenode so that only the internodes are used, these may possibly be dividedso that the explants are approx. 4 mm long. The explants were floated inthe bacterial suspension for 30 min. and blotted dry on a filter paperand transferred to co-cultivation plates (MBa co). The explants werecovered with filter paper moistened in liquid MBa, and the plates werecovered with cloth for 3 days and left at 25° C. The explants were thenwashed in liquid MBa containing 800 mg/l. 2 explants per ml were shakenfor 18 hours, then blotted dry and transferred to selection medium.

The selection medium was solid MBb added 50 mg kanamycin, 800 mgcarbenicillin (Duchefa), 0.1 mg GA₃ (Gibberellic Acid,Sigma) and 1 mgt-Zeatin per litre. The carbenicillin was added to kill any remaining, 4grobacteria.

The explants were subcultivated every 3 weeks.

Regeneration of Whole Potato Plants

Shoots from the explants which by subcultivation was more than 1 cm wereharvested and transferred to a solid MBa medium containing 400 mg/l ofcarbenicillin, 2 μM STS and 0.5 mg/l t-Zeatin. After approx. 2 weeks theshoots were transferred to root-formation medium, that is solid MBa with2 μM STS added. A 5 μM stock of STS was made from 0.19 g of Na₂S₂O₃—5H₂Oand 10.19 mg of AgNO₃ dissolved in 7 ml of water and sterilised byfiltration.

After approx. 2 weeks the shoots had rooted and were ready for plantingin soil.

The plantlets were rinsed in lukewarm water to remove residues of mediaand planted in small pots with TKS 2 instant sphagnum (Flora Gard,Germany). The plantlets were kept moist during the planting and wateredafter. The pots were placed in a “tent” of plastic with 100% humidityand 21-23° C., until the plantlets were rooted in the soil. Then thetent was removed and the plants watered regularly.

After 4 weeks of growth the plants were potted into large pots (diameter27 cm) and transferred to a growth chamber with 16 hours day 22° C. and8 hour night 15° C. When the plants had wiltered down, the tubers wereharvested.

Generation of Microtubers from in vitro Propagated Potato Plants.

From an in vitro propagated plant or a selected shoot a node was cut of5 mm under and 2 mm over the node. The leaf was removed and the explantwas placed on a solid growth medium. The medium contained per litre: 4.4g Murashige and Skoog (MS,(10)) basal salts (Sigma), 60 g sucrose, 2 mgBAP (6-Benzyl-aminopurine, Sigma), 2 g Gelrite (Scott Laboratories,Inc., Carson, Calif.). The explants were incubated for 7 days at 20° C.,with 16 h light/ 8 h dark. The plates were then wrapped in aluminiumfoil and kept in the dark at 20° C. for 21-28 days. Then microtuberscould be harvested, one per explant.

Sprouts from microtubers were generated from the microtubers by cuttingthe tubers in two halves (from top to bottom). They were then placed onsolid MBa medium and incubated in the dark for 7 days at 25° C. and thenewly developed sprouts could be GUS analysed.

Histochemical Localisation of Beta-glucuronidase (GUS) Activity

The tissue was cut in small sections with a razor blade and placed inX-gluc (X-gluc: 5-bromo-4-chloro-3-indolyl-β-glucoronide) is a solutionof 50 mg X-gluc dissolved in a buffer with: 0.1 M NaPO₄ (pH 7.0), 1 mMK₃(Fe(CN)6), 0.1 mM K₄(Fe(CN)6)—3H₂O, 10 mM Na₂EDTA and 3% sucrose (23))solution to cover the section.

Micro tubers were halved, pot grown tubers were sliced into thin slices,leaves were cut into pieces approx. 0.5 cm² and stem tissue was cut intoslices approx. 1 mm thick.

The sections were incubated in X-gluc for 2-12 hours at 37° C. Care wastaken to prevent evaporation. The X-gluc was removed and 96% ethanol wasadded to the tissue sections to extract chlorophyll and other pigments.Incubation in ethanol was overnight at 5° C. and the following day thetissue was transferred to a 2% sucrose solution and after approx. 1 hourexamined in a dissection scope.

Isolation of α-amylase Genomic Clones

Several cDNA clones encoding α-amylase from potato (Solanum tuberosum)had previously been isolated (described in EP-B-0470145). A PstI-SalIfragment from the plasmid pAmyZ3 (EP-B-0470145) encoding a partialα-amylase was used as probe (see “DNA labelling” in Materials andMethods) to screen the genomic potato γ DNA library (see “Constructionof a potato genomic library” in Materials and Methods). Screening ofapprox. 1.6×10⁶ phages was carried out as described in Materials andMethods. Two positive clones were isolated, gPAmy351 and gPAmy331 bythree rounds of plaque purifications.

One clone (gPAmy331) was found to be unstable during isolation of the γDNA (see the method in Materials and Methods), then only the gPAmy351clone was analyzed in details.

A restriction enzyme map of the insert (insert size approx. 22 kb) isshown in FIG. 1.

Mapping of the α-amylase encoding part of the genomic sequence was doneby Southern transfer of various digests of the clone, followed byhybridization with the PstI-SalI fragment of pAmyZ3. gPAmy351 containthe whole promoter region of the α-amylase gene and in addition 1270 bpof the structural gene. This covers the sequences encoding 142 aminoacids corresponding to approx. ⅓ of the total amino acid sequenceencoded by the Amy¾ type potato α-amylase (407 amino acids, seeEP-B-0470145).

Subcloning of the Genomic Fragment Containing the α-amylase Promoter

The EcoR1 fragment of approximately 5.5 kb, indicated by an asterisk inFIG. 1, was subcloned from the gPAmy351 genomic clone into adephosphorylated EcoRI site of a pBS-vector (see Materials and Methods).

This subclone was named Eco 5.5 and it contains the ATG initiation codonand sequences upstream of it (see the next paragraph for a more detaileddescription of the sequence). These upstream sequences will in thefollowing be referred to as the α-Amy 3 promoter.

A large scale plasmid preparation of the Eco 5.5 plasmid was digestedwith EcoRI and HaeIII, this creates a 1350 bp fragment which includesthe ATG initiation codon as well as putative CAAT and TATA boxes.

As shown by others (eg see 24-31) it is often the sequence regioncounting from the ATG initiation codon and approx 1000-1500 bp upstreamthat includes the entire promoter, enough to mediate transcription ofthe gene at the right time and place. The EcoRI-HaeIII fragment wassubcloned into a pBSK-vector, digested with EcoRI and SmaI anddephosphorylated by Alkaline phosphatase (see Materials and Methods).This subclone was named EH8 and the genomic potato fragment it carrieswas chosen for functional analysis. The identity of the insert in theEH8 plasmid was verified by sequencing with T3 and T7 primers (see FIG.2B and Materials and Methods).

Sequencing of the α-amylase Promoter

Approximately 2900 bp of the insert in gPAmy351 (FIG. 1) was sequencedby subcloning various fragments and using the primers shown in FIG. 6(see Materials and Methods). This covers 1734 bp upstream of theinitiation (ATG) codon and 1440 downstream. The sequence map of theregion upstream of the ATG initiation codon is shown in FIG. 2A and theDNA sequence is shown in FIG. 3. This sequence is located near the 3′end of the gPAmy351 insert (see FIG. 1) covering part of the Eco 5.5 kbfragment and the HindIII and EcoRI sites upstream of the initiationcodon, and thereby including the HaeIII-EcoRI (EH) fragment chosen forthe functional analysis.

The α-Amy 3 promoter sequences from the ATG (A in position +1) codon andupstream to position −1734 (see FIG. 3) were compared with the (EMBL)database of published plant sequences (using the PC-gene program fromIntelliGenetics, Inc., California) and also compared with sequences ofall organisms. There were no sequences with significant overall homologyto the α-Amy 3 promoter. A TATA-box is located at position −365.

Comparing the α-Amy 3 promoter with published DNA binding sitessuggested a CAP site to be localized 21 bp downstream of the TATA box(position −344) and two CAAT boxes, one at position −468, which is 103bp upstream of the TATA box, and 124 bp upstream of the CAP site, andthe other at position −557, 192 bp upstream of the TATA box and 213 bpupstream of the CAP site.

The positions of the CAP site, TATA- and CCAAT-box correspond well tothe positions found in other eukaryotic polymerase II promoters (32-33)

α-amylase Promoter Deletions

Plasmid from a large scale preparation of the EH8 subclone was bandedtwice on a CsCl gradient (see Materials and Methods) to obtain puresupercoiled DNA. Running a sample of the plasmid DNA on an agarose gelshowed that at least 85% of the preparation was supercoiled. Then theEH8 plasmid was digested with Bst XI which creates a 3′ overhang andwith BamHI which creates a 5′ overhang end, and care was taken to besure that the digests were complete. An ExoIII/Mung Bean treatment wasdone as described in Materials and Methods, and aliquots were taken at0, 1, 2, 3, 4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 10.5 min. Thedifferent mixtures now containing the nested deletions of the EH8plasmid were used directly in ligation and transformation as explainedin Materials and Methods. A whole range of deletion subclones wereobtained, these are shown in FIG. 4. They were all sequenced with the T3and T7 primers (see FIG. 8) to locate their 5′ end in proportion to theα-Amy 3 promoter. The individual start positions are shown in FIG. 2Awhere the arrows indicate the start and extent of the sequence reactionsof the deleted subclones.

Three of the nested deletion subclones were chosen for functionalanalysis, these were 4-1, 6-15 and 8.5-E (indicated with an asterisk inFIG. 2B).

A large scale plasmid preparation was generated from each of theselected deletions cloned (4-1, 6-15 and 8.5-E) and from the EH8 clone.These were then digested with SacI which cuts in the polylinker of pBSK-and the site blunted (see Materials and Methods) before they weredigested with SalI, which cuts on the opposite site of the promoterinsert.

A pBI101 plasmid (see Materials and Methods) containing a promotorlessβ-glucuronidase (GUS) gene was completely digested with HindIII and theHindIII sites blunted as described in Materials and Methods. Afterwardsthe open plasmid was digested with SalI thereby creating a pBI101HindIII^(Blunt)/SalI vector.

Subcloning and Transformation

The Sac I Blunt/Sal I fragments obtained from the 4-1, 6-15, 8.5-E andEH8 clones were then subcloned into the pBI101 HindIII^(Blunt)/SalIvector and the ligated plasmids transformed into the Agrobacteriumstrain LBA4404 (see Materials and Methods). The colonies obtained weretested using the restriction enzymes PstI and SalI which cut on eitherside of the inserted fragment. Clones which contained an insert of theright size were then analysed by sequencing with a primer named #589(see FIG. 6). The #589 primer primes within the GUS gene of pBI101 andallows the reading of sequences upstream of the promoterless GUS genethereby covering the inserted promoter deletions.

The pBI101 plasmids containing the selected promoter deletions werenamed EH, HFP4, HFP6 and HFP8.

In addition, a Southern transfer of the PstI and SalI digested plasmidswas hybridized with a labelled insert from the EH8 clone which containedthe largest fragment of the α-Amy 3 promoter region, to verify theorigin of the inserts.

To produce a smaller fragment than the one covered by 4-1, 6-15, 8.5-Eor EH8, another subclone was created, the HE subclone. This wasaccomplished by digesting the EH8 subclone with EcoRI and blunt end theEcoRI site followed by a digest of HindIII and then isolate the 288 bpfragment containing 3′ end promoter sequences (see FIG. 2B for positionof the HE fragment). For subcloning this HindIII/EcoRI^(Blunt) fragmentinto a pBI101 vector digested with SmaI and HindIII was used in theligation reaction. The resulting plasmid is called HE and wastransformed into the Agrobacterium strain LBA4404 (see Materials andMethods). The colonies obtained on kanamycin plates were tested bydigesting purified plasmid with the restriction enzymes HindIII andSnaBI. Plasmids from selected colonies were subjected to sequenceanalysis with the #589 primer as explained above.

In total, five deletions of the α-Amy 3 promoter constructs have beenmade as explained in the preceding sections. They cover 1350 bp (EH8),853 bp (HFP4), 672 bp (HFP6), 506 bp (HFP8) and 288 bp (HE) of thesequences upstream of the EcoRI site 5′ to the ATG codon (see FIG. 2 andFIG. 5). They were cloned in front of the promoterless GUS gene of thepBI101 vector (see Materials and Methods).

Transformation of Potato with the Promoter Constructs

Six LBA4404 colonies containing the 5 deletion constructs and thepBI101, were selected and used for transformation of Saturna stem tissueas described in Materials and Methods.

As a negative control some Saturna explants were incubated withnontransformed LBA4404 bacteria and nontransformed shoots obtained fromselection plates without kanamycin. As a positive control some Saturnaexplants were incubated with LBA4404 previously transformed with thepBI121 plasmid. pBI121 contain a GUS cassette controlled by theCauliflower mosaic virus (CaMV) 35S promoter which is constitutivelyexpressed in most plant tissues (34-38).

All regenerated shoots after the first (22 days) and second (49 days)harvest were discarded. After 68 days were 40 shoots from each deletionconstruct, 10 shoot from the negative control and 15 shoots from thepositive control harvested and transferred to root induction medium (seeMaterials and Methods). Each shoots represents, putatively, anindividual transformation event and will in the following be referred toas lines. Each line, if the plant is transformed, represent anindependent transformation event.

Expression of GUS in Leaves of Putatively Transformed Lines

Leaves from the regenerated lines were all GUS tested after rootformation. An expression analysis of the α-amylase genes of the presentinvention revealed that the α-amylase type ¾ is expressed in tuber-,sprout-, stem- and root tissue but no expression was found in leaves.

The GUS testing of leaves, from the putatively transformed deletionlines, from the lines transformed with the pBI101 plasmid, and from thenontransformed control lines, revealed that there were no GUS activityin any of these. In contrast, GUS testing of plants transformed with thepositive control plasmid pBI121 showed GUS expression in the leaves innearly all the plants (see table 1).

Expression of GUS in Microtubers and Sprouts

Micro tubers from the lines described above were generated as describedin Materials and Methods. These microtubers were examined for their GUSactivity and lines containing the EH8, HFP4 and HFP6 deletion constructsshowed positive GUS staining. Again the pBI121 control lines gave GUSpositive microtubers while the lines containing the pBI101 plasmidshowed negative microtubers. Also non-transformed lines microtubersshowed no GUS activity as well as lines transformed with the deletionconstructs HFP8 and HE.

Sprouts generated from microtubers (see Materials and Methods) were alsoGUS analysed and only the lines transformed with the pBI121 (positivecontrol) showed GUS activity.

The results are summarized in Table 1, shown below.

TABLE 1 Expression of GUS Plants Leaf of TD Leaf of Micro- Leaf-likeStem-like pot grown Pot grown with: explant tuber of EP of EP Sproutplant tubers EH  0 (36)* 7 (36) 0 (15) 0 (15) 0 (33) 0 (40) 16 (22) HFP4 0 (36) 2 (36) 0 (15) 0 (15) 0 (31) 0 (40) 15 (24)  HFP6 0 (32) 5(32) 0 (15) 0 (15) 0 (26) 0 (40) 10 (26)  HFPS 0 (30) 0 (30) 0 (10) 0(10) 0 (15) 0 (40) 0 (21) HE 0 (34) 0 (34) 0 (14) 0 (14) 0 (27) 0 (40) 0(23) pBI 101 0 (36) 0 (36) 0 (36) 0 (36) 0 (36) 0 (40) 0 (24) pBI 121 10(15)  10 (15)  10 (10)  10 (10)  6 (6)  10 (15)  5 (5)  Non TD plants 0(15) 0 (9)  — — 0 (9)  0 (15) 0 (12) *The numbers in brackets are thetotal numbers of lines tested. (TD = transformed; EP = Explant)

Expression of GUS in Other Parts of the Planlets

When microtubers are generated they are formed at the end of the stemexplant. On top of the explant most often two leaf-like structures areformed. These stem-like explants and the leaf-like tops were examinedfor GUS activity. As summarized in table 1, none of the deletionscontaining lines showed any GUS activity in either the stem-or leaf-liketissues of the explants.

All the pBI121 lines which showed GUS activity in their microtuber alsohad GUS activity in the stem- and leaf-like structures of the explant.

Expression of GUS in Leaves, Roots, Stems and Tubers of Pot Grown Lines

The regenerated potato lines were also grown in pots in a growth chamber(at 22° C., 16 h light and 15° C., 8 h dark) and leaves, roots, stemsand tubers were GUS analysed. None of the plant lines, except thecontrol lines containing the pBI121 construction, showed GUS activity inthe leaves as summarized in Table 1 and Table 2.

Investigation of GUS expression in tubers harvested from the pot grownplants, repeated the pattern already seen with the lines tested in themicrotubers.

The plants carrying one of three constructs EH8, HFP4 and HFP6 showedGUS activity in the tubers while the plants carrying the HFP8, HE orpBI101 constructs showed no GUS activity in their tubers.

Again the plants carrying the pBI121 construct had GUS activity in theirpot grown tubers as expected.

A GUS analysis of the lines listed in table 2 (except for the positivecontrol plants carrying pBI121) showed that there were no GUS activityfound in root, stem or leaf tissues even though the lines EH8, HFP4 andHFP6 containing lines clearly showed GUS activity in both microtuber andpot grown tubers.

TABLE 2 Expression of GUS in pot grown potatoes Line No. Construct StemLeaf Root Tuber Saturna control — 0 0 0 0 K702-15.2 EH8 0 0 0 +K702-41.6 0 0 0 + K702-47.3 0 0 0 + K702-28.2 0 0 0 + K699-2.2 HFP4 0 00 + K699-31.5 0 0 0 + K699-44.2 0 0 0 + K700-1.5 HFP6 0 0 0 + K700-24.30 0 0 + K700-38.2 0 0 0 + K703-44.6 HFP8 0 0 0 0 K701-5.3 HE 0 0 0 0K701-15.2 0 0 0 0 K701-18.2 0 0 0 0 K701-16.2 0 0 0 0 K701-49.2 0 0 0 0K661-10.4 pBI121 + + + + K661-15.3 + + + +

In conclusion, it is clear that none of the α-Amy 3 promoter deletionconstructs covering 1534 bp upstream of the ATG initiation codon leadsto expression of an otherwise promoterless GUS gene in leaves ofplantlcts or pot grown plants, in leaf-and stem-like tissues ofmicrotuber explants or in roots and stems of pot grown plants.

GUS expression is only found in microtubers and pot grown tubers clearlyshowing that this α-Amy 3 promoter contains a tuber specific elementclearly separable from the stem, sprout and root expressing element(s)situated upstream of the EH8 deletions 5′ end.

In addition this invention also shows that the tuber specific element issituated near and upstream of the HFPS deletions 5′ end and is coveredby the delta sequence. This invention also show that the stem, sproutand root expressing element(s) is positioned upstream of the 5′ end ofEH8, since neither EH8, HFP4, HFP6, HFP8 nor EH constructs gave GUSexpression in these tissues.

It is therefore concluded that the elements directing root-specific,stem-specific and sprout-specific expression are located far upstream inthe 351 promoter.

The applicability of the promoters is widespread. With the promoters itis possible to direct the expression of proteins into different tissuesin the potato plant. It is even possible to direct the expression ofproteins into different tissues in othe r dicot plants.

pJK4

The potato α-amylase encoding sequences originate from plasmid pAmyZ4(see detailed description in EP-B-0470145). Briefly pAmyZ4 encodes a 407amino acid long potato α-amylase precursor and in addition contains 149bp 5′ and 201 bp 3′ untranslated sequences positioned in the EcoRI siteof the plasmid pBSK-'s polylinker.

The antisense α-amylase construction pJK4, containing the sequence shownas SEQ. I.D. No. 19, was made by using the SacI and EcoRV fragment frompAMYZ4 and subcloning it into an appropriate plasmid—such as SmaI andSacI digested pEPL plasmid (see FIG. 10). This places the antisensesequence downstream of an enhanced 35S promoter (E35S) and upstream ofthe DW2t terminator. This plasmid is called pEPLZ4Sac-Eco and a partialHindIII fragment containing the E35S promoter, the antisense potatosequence and the DW2t terminator was further subcloned into a HindIIIdigested pBI121, thereby creating the binary plasmid pJK4, see FIG. 9.

Other modifications of the present invention will be apparent to thoseskilled in the art without departing from the scope of the invention.

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27 166 base pairs nucleic acid double linear Genomic DNA unknown 1ATAGCTTGAG GCGAAAATAT TTAATAAAAA CACTTCTTAA TTTGTTTATA TGTTCAATTG 60AACATGTCCG TGATTAGAAA ATTAAATTAA ATTCAATGAC AAATTTAATA ATTTGACACA 120AAATTTATGA AAAAAATATC AAAATATAAA GAAATATTTT TTTTGA 166 291 base pairsnucleic acid double linear DNA (genomic) unknown 2 AAGCTTCCAA TGAACCGTTGCCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 60 CTGACGAACG ATTTCGGAGCGGAACGAATC CAGAAAATGG ATTACTTTCT ATAAATTCCT 120 CGAATCTCAA CTCCATTTCGTAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 180 TTTTGTATTT CTGGTTTATGTGGTGATCGA ATTTTCAATT TTTTTACTGG TAGTGATTCC 240 TACTTTTCTT CAATTGCATTTCTCCTTTTT CCATTTCACG GTTGAGAATT C 291 508 base pairs nucleic aciddouble linear DNA (genomic) unknown 3 AATGGATTAA AAAGAAAAAA AAAACAAATAAATTGAACCG GGATAAGTTG GTTGTTTAAT 60 TGATTATTGA TTATGATCTC AATTTGACATTTTGCGCGAT CTTTCGACCT CAATTCGTAT 120 GAACTGACAC TACGCCAATG GACAGTCGCCGTCGTCACCG CCACCGCACT ATTCTCGACG 180 CGTCGTCTAT CTCCTCCACC CCACAGCCGTCAATTCCAAG CTTCCAATGA ACCGTTGCCA 240 TGTGTCACTG CCTATTCACC GCGAAACATGAATATCACTG ACGAACGATT TCGGAGCGGA 300 ACGAATCCAG AAAATGGATT ACTTTCTATAAATTCCTCGA ATCTCAACTC CATTTCGTAA 360 AAATAAAATT AAAAATATTG TTTCTTTTTGTATTTCTTTT TGTATTTCTG GTTTATGTGG 420 TGATCGAATT TTCAATTTTT TTACTGGTAGTGATTCCTAC TTTTCTTCAA TTGCATTTCT 480 CCTTTTTCCA TTTCACGGTT GAGAATTC 508514 base pairs nucleic acid double linear DNA (genomic) unknown 4TTTTGAAATG GATTAAAAAG AAAAAAAAAA CAAATAAATT GAACCGGGAT AAGTTGGTTG 60TTTAATTGAT TATTGATTAT GATCTCAATT TGACATTTTG CGCGATCTTT CGACCTCAAT 120TCGTATGAAC TGACACTACG CCAATGGACA GTCGCCGTCG TCACCGCCAC CGCACTATTC 180TCGACGCGTC GTCTATCTCC TCCACCCCAC AGCCGTCAAT TCCAAGCTTC CAATGAACCG 240TTGCCATGTG TCACTGCCTA TTCACCGCGA AACATGAATA TCACTGACGA ACGATTTCGG 300AGCGGAACGA ATCCAGAAAA TGGATTACTT TCTATAAATT CCTCGAATCT CAACTCCATT 360TCGTAAAAAT AAAATTAAAA ATATTGTTTC TTTTTGTATT TCTTTTTGTA TTTCTGGTTT 420ATGTGGTGAT CGAATTTTCA ATTTTTTTAC TGGTAGTGAT TCCTACTTTT CTTCAATTGC 480ATTTCTCCTT TTTCCATTTC ACGGTTGAGA ATTC 514 518 base pairs nucleic aciddouble linear DNA (genomic) unknown 5 TTTTTTTTGA AATGGATTAA AAAGAAAAAAAAAACAAATA AATTGAACCG GGATAAGTTG 60 GTTGTTTAAT TGATTATTGA TTATGATCTCAATTTGACAT TTTGCGCGAT CTTTCGACCT 120 CAATTCGTAT GAACTGACAC TACGCCAATGGACAGTCGCC GTCGTCACCG CCACCGCACT 180 ATTCTCGACG CGTCGTCTAT CTCCTCCACCCCACAGCCGT CAATTCCAAG CTTCCAATGA 240 ACCGTTGCCA TGTGTCACTG CCTATTCACCGCGAAACATG AATATCACTG ACGAACGATT 300 TCGGAGCGGA ACGAATCCAG AAAATGGATTACTTTCTATA AATTCCTCGA ATCTCAACTC 360 CATTTCGTAA AAATAAAATT AAAAATATTGTTTCTTTTTG TATTTCTTTT TGTATTTCTG 420 GTTTATGTGG TGATCGAATT TTCAATTTTTTTACTGGTAG TGATTCCTAC TTTTCTTCAA 480 TTGCATTTCT CCTTTTTCCA TTTCACGGTTGAGAATTC 518 631 base pairs nucleic acid double linear DNA (genomic)unknown 6 GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT AAATTAAATTCAATGACAAA 60 TTTAATAATT TGACACAAAA TTTATGAAAA AAATATCAAA ATATAAAGAAATATTTTTTT 120 TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTGAA CCGGGATAAGTTGGTTGTTT 180 AATTGATTAT TGATTATGAT CTCAATTTGA CATTTTGCGC GATCTTTCGACCTCAATTCG 240 TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA CCGCCACCGCACTATTCTCG 300 ACGCGTCGTC TATCTCCTCC ACCCCACAGC CGTCAATTCC AAGCTTCCAATGAACCGTTG 360 CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA CTGACGAACGATTTCGGAGC 420 GGAACGAATC CAGAAAATGG ATTACTTTCT ATAAATTCCT CGAATCTCAACTCCATTTCG 480 TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT TTTTGTATTTCTGGTTTATG 540 TGGTGATCGA ATTTTCAATT TTTTTACTGG TAGTGATTCC TACTTTTCTTCAATTGCATT 600 TCTCCTTTTT CCATTTCACG GTTGAGAATT C 631 674 base pairsnucleic acid double linear DNA (genomic) unknown 7 ATAGCTTGAG GCGAAAATATTTAATAAAAA CACTTCTTAA TTTGTTTATA TGTTCAATTG 60 AACATGTCCG TGATTAGAAAATTAAATTAA ATTCAATGAC AAATTTAATA ATTTGACACA 120 AAATTTATGA AAAAAATATCAAAATATAAA GAAATATTTT TTTTGAAATG GATTAAAAAG 180 AAAAAAAAAA CAAATAAATTGAACCGGGAT AAGTTGGTTG TTTAATTGAT TATTGATTAT 240 GATCTCAATT TGACATTTTGCGCGATCTTT CGACCTCAAT TCGTATGAAC TGACACTACG 300 CCAATGGACA GTCGCCGTCGTCACCGCCAC CGCACTATTC TCGACGCGTC GTCTATCTCC 360 TCCACCCCAC AGCCGTCAATTCCAAGCTTC CAATGAACCG TTGCCATGTG TCACTGCCTA 420 TTCACCGCGA AACATGAATATCACTGACGA ACGATTTCGG AGCGGAACGA ATCCAGAAAA 480 TGGATTACTT TCTATAAATTCCTCGAATCT CAACTCCATT TCGTAAAAAT AAAATTAAAA 540 ATATTGTTTC TTTTTGTATTTCTTTTTGTA TTTCTGGTTT ATGTGGTGAT CGAATTTTCA 600 ATTTTTTTAC TGGTAGTGATTCCTACTTTT CTTCAATTGC ATTTCTCCTT TTTCCATTTC 660 ACGGTTGAGA ATTC 674 687base pairs nucleic acid double linear DNA (genomic) unknown 8 TTCCTTTCCTCATATAGCTT GAGGCGAAAA TATTTAATAA AAACACTTCT TAATTTGTTT 60 ATATGTTCAATTGAACATGT CCGTGATTAG AAAATTAAAT TAAATTCAAT GACAAATTTA 120 ATAATTTGACACAAAATTTA TGAAAAAAAT ATCAAAATAT AAAGAAATAT TTTTTTTGAA 180 ATGGATTAAAAAGAAAAAAA AAACAAATAA ATTGAACCGG GATAAGTTGG TTGTTTAATT 240 GATTATTGATTATGATCTCA ATTTGACATT TTGCGCGATC TTTCGACCTC AATTCGTATG 300 AACTGACACTACGCCAATGG ACAGTCGCCG TCGTCACCGC CACCGCACTA TTCTCGACGC 360 GTCGTCTATCTCCTCCACCC CACAGCCGTC AATTCCAAGC TTCCAATGAA CCGTTGCCAT 420 GTGTCACTGCCTATTCACCG CGAAACATGA ATATCACTGA CGAACGATTT CGGAGCGGAA 480 CGAATCCAGAAAATGGATTA CTTTCTATAA ATTCCTCGAA TCTCAACTCC ATTTCGTAAA 540 AATAAAATTAAAAATATTGT TTCTTTTTGT ATTTCTTTTT GTATTTCTGG TTTATGTGGT 600 GATCGAATTTTCAATTTTTT TACTGGTAGT GATTCCTACT TTTCTTCAAT TGCATTTCTC 660 CTTTTTCCATTTCACGGTTG AGAATTC 687 693 base pairs nucleic acid double linear DNA(genomic) unknown 9 CACTGATTCC TTTCCTCATA TAGCTTGAGG CGAAAATATTTAATAAAAAC ACTTCTTAAT 60 TTGTTTATAT GTTCAATTGA ACATGTCCGT GATTAGAAAATTAAATTAAA TTCAATGACA 120 AATTTAATAA TTTGACACAA AATTTATGAA AAAAATATCAAAATATAAAG AAATATTTTT 180 TTTGAAATGG ATTAAAAAGA AAAAAAAAAC AAATAAATTGAACCGGGATA AGTTGGTTGT 240 TTAATTGATT ATTGATTATG ATCTCAATTT GACATTTTGCGCGATCTTTC GACCTCAATT 300 CGTATGAACT GACACTACGC CAATGGACAG TCGCCGTCGTCACCGCCACC GCACTATTCT 360 CGACGCGTCG TCTATCTCCT CCACCCCACA GCCGTCAATTCCAAGCTTCC AATGAACCGT 420 TGCCATGTGT CACTGCCTAT TCACCGCGAA ACATGAATATCACTGACGAA CGATTTCGGA 480 GCGGAACGAA TCCAGAAAAT GGATTACTTT CTATAAATTCCTCGAATCTC AACTCCATTT 540 CGTAAAAATA AAATTAAAAA TATTGTTTCT TTTTGTATTTCTTTTTGTAT TTCTGGTTTA 600 TGTGGTGATC GAATTTTCAA TTTTTTTACT GGTAGTGATTCCTACTTTTC TTCAATTGCA 660 TTTCTCCTTT TTCCATTTCA CGGTTGAGAA TTC 693 758base pairs nucleic acid double linear DNA (genomic) unknown 10CTTGCGCCTT TCCCTAAATT AAGTAAAACT CTTCGCCTCA TGCCTTACGC CTCCGCCTTT 60TAAAACACTG ATTCCTTTCC TCATATAGCT TGAGGCGAAA ATATTTAATA AAAACACTTC 120TTAATTTGTT TATATGTTCA ATTGAACATG TCCGTGATTA GAAAATTAAA TTAAATTCAA 180TGACAAATTT AATAATTTGA CACAAAATTT ATGAAAAAAA TATCAAAATA TAAAGAAATA 240TTTTTTTTGA AATGGATTAA AAAGAAAAAA AAAACAAATA AATTGAACCG GGATAAGTTG 300GTTGTTTAAT TGATTATTGA TTATGATCTC AATTTGACAT TTTGCGCGAT CTTTCGACCT 360CAATTCGTAT GAACTGACAC TACGCCAATG GACAGTCGCC GTCGTCACCG CCACCGCACT 420ATTCTCGACG CGTCGTCTAT CTCCTCCACC CCACAGCCGT CAATTCCAAG CTTCCAATGA 480ACCGTTGCCA TGTGTCACTG CCTATTCACC GCGAAACATG AATATCACTG ACGAACGATT 540TCGGAGCGGA ACGAATCCAG AAAATGGATT ACTTTCTATA AATTCCTCGA ATCTCAACTC 600CATTTCGTAA AAATAAAATT AAAAATATTG TTTCTTTTTG TATTTCTTTT TGTATTTCTG 660GTTTATGTGG TGATCGAATT TTCAATTTTT TTACTGGTAG TGATTCCTAC TTTTCTTCAA 720TTGCATTTCT CCTTTTTCCA TTTCACGGTT GAGAATTC 758 855 base pairs nucleicacid double linear DNA (genomic) unknown 11 CAAATTTTGA TGTATTTTTATAATTTTGTA TTATTATATT ATTATACTAT ATTTAAAAAT 60 TTAAAGATCC ATAGGGCTTACGCCCCACGT CAAGAGGCTT GCGCCTTTCC CTAAATTAAG 120 TAAAACTCTT CGCCTCATGCCTTACGCCTC CGCCTTTTAA AACACTGATT CCTTTCCTCA 180 TATAGCTTGA GGCGAAAATATTTAATAAAA ACACTTCTTA ATTTGTTTAT ATGTTCAATT 240 GAACATGTCC GTGATTAGAAAATTAAATTA AATTCAATGA CAAATTTAAT AATTTGACAC 300 AAAATTTATG AAAAAAATATCAAAATATAA AGAAATATTT TTTTTGAAAT GGATTAAAAA 360 GAAAAAAAAA ACAAATAAATTGAACCGGGA TAAGTTGGTT GTTTAATTGA TTATTGATTA 420 TGATCTCAAT TTGACATTTTGCGCGATCTT TCGACCTCAA TTCGTATGAA CTGACACTAC 480 GCCAATGGAC AGTCGCCGTCGTCACCGCCA CCGCACTATT CTCGACGCGT CGTCTATCTC 540 CTCCACCCCA CAGCCGTCAATTCCAAGCTT CCAATGAACC GTTGCCATGT GTCACTGCCT 600 ATTCACCGCG AAACATGAATATCACTGACG AACGATTTCG GAGCGGAACG AATCCAGAAA 660 ATGGATTACT TTCTATAAATTCCTCGAATC TCAACTCCAT TTCGTAAAAA TAAAATTAAA 720 AATATTGTTT CTTTTTGTATTTCTTTTTGT ATTTCTGGTT TATGTGGTGA TCGAATTTTC 780 AATTTTTTTA CTGGTAGTGATTCCTACTTT TCTTCAATTG CATTTCTCCT TTTTCCATTT 840 CACGGTTGAG AATTC 855 859base pairs nucleic acid double linear DNA (genomic) unknown 12ATTTCAAATT TTGATGTATT TTTATAATTT TGTATTATTA TATTATTATA CTATATTTAA 60AAATTTAAAG ATCCATAGGG CTTACGCCCC ACGTCAAGAG GCTTGCGCCT TTCCCTAAAT 120TAAGTAAAAC TCTTCGCCTC ATGCCTTACG CCTCCGCCTT TTAAAACACT GATTCCTTTC 180CTCATATAGC TTGAGGCGAA AATATTTAAT AAAAACACTT CTTAATTTGT TTATATGTTC 240AATTGAACAT GTCCGTGATT AGAAAATTAA ATTAAATTCA ATGACAAATT TAATAATTTG 300ACACAAAATT TATGAAAAAA ATATCAAAAT ATAAAGAAAT ATTTTTTTTG AAATGGATTA 360AAAAGAAAAA AAAAACAAAT AAATTGAACC GGGATAAGTT GGTTGTTTAA TTGATTATTG 420ATTATGATCT CAATTTGACA TTTTGCGCGA TCTTTCGACC TCAATTCGTA TGAACTGACA 480CTACGCCAAT GGACAGTCGC CGTCGTCACC GCCACCGCAC TATTCTCGAC GCGTCGTCTA 540TCTCCTCCAC CCCACAGCCG TCAATTCCAA GCTTCCAATG AACCGTTGCC ATGTGTCACT 600GCCTATTCAC CGCGAAACAT GAATATCACT GACGAACGAT TTCGGAGCGG AACGAATCCA 660GAAAATGGAT TACTTTCTAT AAATTCCTCG AATCTCAACT CCATTTCGTA AAAATAAAAT 720TAAAAATATT GTTTCTTTTT GTATTTCTTT TTGTATTTCT GGTTTATGTG GTGATCGAAT 780TTTCAATTTT TTTACTGGTA GTGATTCCTA CTTTTCTTCA ATTGCATTTC TCCTTTTTCC 840ATTTCACGGT TGAGAATTC 859 1214 base pairs nucleic acid double linear DNA(genomic) unknown 13 GAAGGTGATT ATACATTACG TAACATTTCT TTTAAAAATATGTAAGCAAA TTTACTTTTT 60 AACTTATCAT TGATCTTCAT GGTTTTGTCA TAAATCTCAAAGTTATCATA TTTTATATAG 120 CTATTTGAAA GTAATTTTAT TTTTACTCAT CATTGAGTGATGCTTTTATT ATAATACTAG 180 TAAGTTTTAT TTATTATTTT CTTTTAGGGG TGAATTGTATAATATAATAA AAAATATATT 240 TTTAGAAATA ATGATTCTTT TATTATTAAA AAGTTAAGATATTAGATTAT TTATGCTTGT 300 ATAATAATGA ACGAAGTTTT ATTTTCTATG AGTTTCATTAATCATGTTTG TAATTATTTC 360 AAATTTTGAT GTATTTTTAT AATTTTGTAT TATTATATTATTATACTATA TTTAAAAATT 420 TAAAGATCCA TAGGGCTTAC GCCCCACGTC AAGAGGCTTGCGCCTTTCCC TAAATTAAGT 480 AAAACTCTTC GCCTCATGCC TTACGCCTCC GCCTTTTAAAACACTGATTC CTTTCCTCAT 540 ATAGCTTGAG GCGAAAATAT TTAATAAAAA CACTTCTTAATTTGTTTATA TGTTCAATTG 600 AACATGTCCG TGATTAGAAA ATTAAATTAA ATTCAATGACAAATTTAATA ATTTGACACA 660 AAATTTATGA AAAAAATATC AAAATATAAA GAAATATTTTTTTTGAAATG GATTAAAAAG 720 AAAAAAAAAA CAAATAAATT GAACCGGGAT AAGTTGGTTGTTTAATTGAT TATTGATTAT 780 GATCTCAATT TGACATTTTG CGCGATCTTT CGACCTCAATTCGTATGAAC TGACACTACG 840 CCAATGGACA GTCGCCGTCG TCACCGCCAC CGCACTATTCTCGACGCGTC GTCTATCTCC 900 TCCACCCCAC AGCCGTCAAT TCCAAGCTTC CAATGAACCGTTGCCATGTG TCACTGCCTA 960 TTCACCGCGA AACATGAATA TCACTGACGA ACGATTTCGGAGCGGAACGA ATCCAGAAAA 1020 TGGATTACTT TCTATAAATT CCTCGAATCT CAACTCCATTTCGTAAAAAT AAAATTAAAA 1080 ATATTGTTTC TTTTTGTATT TCTTTTTGTA TTTCTGGTTTATGTGGTGAT CGAATTTTCA 1140 ATTTTTTTAC TGGTAGTGAT TCCTACTTTT CTTCAATTGCATTTCTCCTT TTTCCATTTC 1200 ACGGTTGAGA ATTC 1214 1232 base pairs nucleicacid double linear DNA (genomic) unknown 14 ACTATTTGAT AACATTATGAAGGTGATTAT ACATTACGTA ACATTTCTTT TAAAAATATG 60 TAAGCAAATT TACTTTTTAACTTATCATTG ATCTTCATGG TTTTGTCATA AATCTCAAAG 120 TTATCATATT TTATATAGCTATTTGAAAGT AATTTTATTT TTACTCATCA TTGAGTGATG 180 CTTTTATTAT AATACTAGTAAGTTTTATTT ATTATTTTCT TTTAGGGGTG AATTGTATAA 240 TATAATAAAA AATATATTTTTAGAAATAAT GATTCTTTTA TTATTAAAAA GTTAAGATAT 300 TAGATTATTT ATGCTTGTATAATAATGAAC GAAGTTTTAT TTTCTATGAG TTTCATTAAT 360 CATGTTTGTA ATTATTTCAAATTTTGATGT ATTTTTATAA TTTTGTATTA TTATATTATT 420 ATACTATATT TAAAAATTTAAAGATCCATA GGGCTTACGC CCCACGTCAA GAGGCTTGCG 480 CCTTTCCCTA AATTAAGTAAAACTCTTCGC CTCATGCCTT ACGCCTCCGC CTTTTAAAAC 540 ACTGATTCCT TTCCTCATATAGCTTGAGGC GAAAATATTT AATAAAAACA CTTCTTAATT 600 TGTTTATATG TTCAATTGAACATGTCCGTG ATTAGAAAAT TAAATTAAAT TCAATGACAA 660 ATTTAATAAT TTGACACAAAATTTATGAAA AAAATATCAA AATATAAAGA AATATTTTTT 720 TTGAAATGGA TTAAAAAGAAAAAAAAAACA AATAAATTGA ACCGGGATAA GTTGGTTGTT 780 TAATTGATTA TTGATTATGATCTCAATTTG ACATTTTGCG CGATCTTTCG ACCTCAATTC 840 GTATGAACTG ACACTACGCCAATGGACAGT CGCCGTCGTC ACCGCCACCG CACTATTCTC 900 GACGCGTCGT CTATCTCCTCCACCCCACAG CCGTCAATTC CAAGCTTCCA ATGAACCGTT 960 GCCATGTGTC ACTGCCTATTCACCGCGAAA CATGAATATC ACTGACGAAC GATTTCGGAG 1020 CGGAACGAAT CCAGAAAATGGATTACTTTC TATAAATTCC TCGAATCTCA ACTCCATTTC 1080 GTAAAAATAA AATTAAAAATATTGTTTCTT TTTGTATTTC TTTTTGTATT TCTGGTTTAT 1140 GTGGTGATCG AATTTTCAATTTTTTTACTG GTAGTGATTC CTACTTTTCT TCAATTGCAT 1200 TTCTCCTTTT TCCATTTCACGGTTGAGAAT TC 1232 1352 base pairs nucleic acid double linear DNA(genomic) unknown 15 GGCCTCACAT CAACCTTCAT AATTCTTGAA TGAATGAATGATAGACTTCA TAATTTTTTA 60 ACCTATACAT ATAAGAAAAT TGAGAGTAAC TCAAATAACAAGTTGTAGTA TCACATCTTT 120 ACTATTTGAT AACATTATGA AGGTGATTAT ACATTACGTAACATTTCTTT TAAAAATATG 180 TAAGCAAATT TACTTTTTAA CTTATCATTG ATCTTCATGGTTTTGTCATA AATCTCAAAG 240 TTATCATATT TTATATAGCT ATTTGAAAGT AATTTTATTTTTACTCATCA TTGAGTGATG 300 CTTTTATTAT AATACTAGTA AGTTTTATTT ATTATTTTCTTTTAGGGGTG AATTGTATAA 360 TATAATAAAA AATATATTTT TAGAAATAAT GATTCTTTTATTATTAAAAA GTTAAGATAT 420 TAGATTATTT ATGCTTGTAT AATAATGAAC GAAGTTTTATTTTCTATGAG TTTCATTAAT 480 CATGTTTGTA ATTATTTCAA ATTTTGATGT ATTTTTATAATTTTGTATTA TTATATTATT 540 ATACTATATT TAAAAATTTA AAGATCCATA GGGCTTACGCCCCACGTCAA GAGGCTTGCG 600 CCTTTCCCTA AATTAAGTAA AACTCTTCGC CTCATGCCTTACGCCTCCGC CTTTTAAAAC 660 ACTGATTCCT TTCCTCATAT AGCTTGAGGC GAAAATATTTAATAAAAACA CTTCTTAATT 720 TGTTTATATG TTCAATTGAA CATGTCCGTG ATTAGAAAATTAAATTAAAT TCAATGACAA 780 ATTTAATAAT TTGACACAAA ATTTATGAAA AAAATATCAAAATATAAAGA AATATTTTTT 840 TTGAAATGGA TTAAAAAGAA AAAAAAAACA AATAAATTGAACCGGGATAA GTTGGTTGTT 900 TAATTGATTA TTGATTATGA TCTCAATTTG ACATTTTGCGCGATCTTTCG ACCTCAATTC 960 GTATGAACTG ACACTACGCC AATGGACAGT CGCCGTCGTCACCGCCACCG CACTATTCTC 1020 GACGCGTCGT CTATCTCCTC CACCCCACAG CCGTCAATTCCAAGCTTCCA ATGAACCGTT 1080 GCCATGTGTC ACTGCCTATT CACCGCGAAA CATGAATATCACTGACGAAC GATTTCGGAG 1140 CGGAACGAAT CCAGAAAATG GATTACTTTC TATAAATTCCTCGAATCTCA ACTCCATTTC 1200 GTAAAAATAA AATTAAAAAT ATTGTTTCTT TTTGTATTTCTTTTTGTATT TCTGGTTTAT 1260 GTGGTGATCG AATTTTCAAT TTTTTTACTG GTAGTGATTCCTACTTTTCT TCAATTGCAT 1320 TTCTCCTTTT TCCATTTCAC GGTTGAGAAT TC 1352 1734base pairs nucleic acid double linear DNA (genomic) unknown 16TCTTTAAGTT GTTTGCTTGA TTTTTCTTCT TCAATCTTCT ATATTTAATT CGTTTTAGCT 60TCAAACTTCT TCAATTTTAT TTCAATTTAA TTCTACAAAA AAAATCTCTA TTTAGCACCA 120TTCATAAAAT TCATGCTCAA AATGGGCAAA CATAAATAAT AAATGTGAAG TAAATAATGG 180ATTAAAATAT ATATTTTTGG GCCTCACATC AACCTTCATA ATTCTTGAAT GAATGAATGA 240TAGACTTCAT AATTTTTTAA CCTATACATA TAAGAAAATT GAGAGTAACT CAAATAACAA 300GTTGTAGTAT CACATCTTTA CTATTTGATA ACATTATGAA GGTGATTATA CATTACGTAA 360CATTTCTTTT AAAAATATGT AAGCAAATTT ACTTTTTAAC TTATCATTGA TCTTCATGGT 420TTTGTCATAA ATCTCAAAGT TATCATATTT TATATAGCTA TTTGAAAGTA ATTTTATTTT 480TACTCATCAT TGAGTGATGC TTTTATTATA ATACTAGTAA GTTTTATTTA TTATTTTCTT 540TTAGGGGTGA ATTGTATAAT ATAATAAAAA ATATATTTTT AGAAATAATG ATTCTTTTAT 600TATTAAAAAG TTAAGATATT AGATTATTTA TGCTTGTATA ATAATGAACG AAGTTTTATT 660TTCTATGAGT TTCATTAATC ATGTTTGTAA TTATTTCAAA TTTTGATGTA TTTTTATAAT 720TTTGTATTAT TATATTATTA TACTATATTT AAAAATTTAA AGATCCATAG GGCTTACGCC 780CCACGTCAAG AGGCTTGCGC CTTTCCCTAA ATTAAGTAAA ACTCTTCGCC TCATGCCTTA 840CGCCTCCGCC TTTTAAAACA CTGATTCCTT TCCTCATATA GCTTGAGGCG AAAATATTTA 900ATAAAAACAC TTCTTAATTT GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 960AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA TTTATGAAAA AAATATCAAA 1020ATATAAAGAA ATATTTTTTT TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTGAA 1080CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT CTCAATTTGA CATTTTGCGC 1140GATCTTTCGA CCTCAATTCG TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA 1200CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC ACCCCACAGC CGTCAATTCC 1260AAGCTTCCAA TGAACCGTTG CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 1320CTGACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG ATTACTTTCT ATAAATTCCT 1380CGAATCTCAA CTCCATTTCG TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 1440TTTTGTATTT CTGGTTTATG TGGTGATCGA ATTTTCAATT TTTTTACTGG TAGTGATTCC 1500TACTTTTCTT CAATTGCATT TCTCCTTTTT CCATTTCACG GTTGAGAATT CATGATTCCT 1560TATCAGAGGA ATCGATCCGA TTTGACTAAT TTCACTTTTC GTCTGTATAA ATACCAGAGT 1620ATCTAGGTTG AGGAACGTAA TTTCAAGCTG CGATCGGCTT TTTCCCCTGA ACGAGCAAAC 1680ACAGGTTGTG GGTTCGAGTT AGCAAGGGAC GTATAATCTC AACTACAATC CATT 1734 1920base pairs nucleic acid double linear DNA (genomic) unknown 17TCTTTAAGTT GTTTGCTTGA TTTTTCTTCT TCAATCTTCT ATATTTAATT CGTTTTAGCT 60TCAAACTTCT TCAATTTTAT TTCAATTTAA TTCTACAAAA AAAATCTCTA TTTAGCACCA 120TTCATAAAAT TCATGCTCAA AATGGGCAAA CATAAATAAT AAATGTGAAG TAAATAATGG 180ATTAAAATAT ATATTTTTGG GCCTCACATC AACCTTCATA ATTCTTGAAT GAATGAATGA 240TAGACTTCAT AATTTTTTAA CCTATACATA TAAGAAAATT GAGAGTAACT CAAATAACAA 300GTTGTAGTAT CACATCTTTA CTATTTGATA ACATTATGAA GGTGATTATA CATTACGTAA 360CATTTCTTTT AAAAATATGT AAGCAAATTT ACTTTTTAAC TTATCATTGA TCTTCATGGT 420TTTGTCATAA ATCTCAAAGT TATCATATTT TATATAGCTA TTTGAAAGTA ATTTTATTTT 480TACTCATCAT TGAGTGATGC TTTTATTATA ATACTAGTAA GTTTTATTTA TTATTTTCTT 540TTAGGGGTGA ATTGTATAAT ATAATAAAAA ATATATTTTT AGAAATAATG ATTCTTTTAT 600TATTAAAAAG TTAAGATATT AGATTATTTA TGCTTGTATA ATAATGAACG AAGTTTTATT 660TTCTATGAGT TTCATTAATC ATGTTTGTAA TTATTTCAAA TTTTGATGTA TTTTTATAAT 720TTTGTATTAT TATATTATTA TACTATATTT AAAAATTTAA AGATCCATAG GGCTTACGCC 780CCACGTCAAG AGGCTTGCGC CTTTCCCTAA ATTAAGTAAA ACTCTTCGCC TCATGCCTTA 840CGCCTCCGCC TTTTAAAACA CTGATTCCTT TCCTCATATA GCTTGAGGCG AAAATATTTA 900ATAAAAACAC TTCTTAATTT GTTTATATGT TCAATTGAAC ATGTCCGTGA TTAGAAAATT 960AAATTAAATT CAATGACAAA TTTAATAATT TGACACAAAA TTTATGAAAA AAATATCAAA 1020ATATAAAGAA ATATTTTTTT TGAAATGGAT TAAAAAGAAA AAAAAAACAA ATAAATTGAA 1080CCGGGATAAG TTGGTTGTTT AATTGATTAT TGATTATGAT CTCAATTTGA CATTTTGCGC 1140GATCTTTCGA CCTCAATTCG TATGAACTGA CACTACGCCA ATGGACAGTC GCCGTCGTCA 1200CCGCCACCGC ACTATTCTCG ACGCGTCGTC TATCTCCTCC ACCCCACAGC CGTCAATTCC 1260AAGCTTCCAA TGAACCGTTG CCATGTGTCA CTGCCTATTC ACCGCGAAAC ATGAATATCA 1320CTGACGAACG ATTTCGGAGC GGAACGAATC CAGAAAATGG ATTACTTTCT ATAAATTCCT 1380CGAATCTCAA CTCCATTTCG TAAAAATAAA ATTAAAAATA TTGTTTCTTT TTGTATTTCT 1440TTTTGTATTT CTGGTTTATG TGGTGATCGA ATTTTCAATT TTTTTACTGG TAGTGATTCC 1500TACTTTTCTT CAATTGCATT TCTCCTTTTT CCATTTCACG GTTGAGAATT CATGATTCCT 1560TATCAGAGGA ATCGATCCGA TTTGACTAAT TTCACTTTTC GTCTGTATAA ATACCAGAGT 1620ATCTAGGTTG AGGAACGTAA TTTCAAGCTG CGATCGGCTT TTTCCCCTGA ACGAGCAAAC 1680ACAGGTTGTG GGTTCGAGTT AGCAAGGGAC GTATAATCTC AACTACAATC CATTATGGCG 1740CTTGATGAAA GTCAGCAGTC TGATCCATGT AAGGTTCTCT TTTCCTTTAT ATATGCTTCA 1800TAATTGAGAA GGAAGACGGA GATTTGAACT TAATAAAGGC GAAGATTTGA ACAAAATATT 1860TTGGTATTTC ATTTAAAACT TTACCAGTTC TAAGAGTAAA TGATTGGGAT GTGCATGTCC 19201570 base pairs nucleic acid double linear DNA (genomic) unknown 18TGTGGTGATC GAATTTTCAA TTTTTTTACT GAGTATCTAG GTTGAGGAAC GTAATTTCAA 60GCTGCGATCG GCTTTTTCCC CTGAACGAGC AAACACAGGT TGTGGGTTCG AGTTAGCAAG 120GGACGTATAA TCTCAACTAC AATCCATTAT GGCGCTTGAT GAAAGTCAGC AGTCTGATCC 180ATTGGTTGTG ATACGCAATG GAAAGGAGAT CATATTGCAG GCATTCGACT GGGAATCTCA 240TAAACATGAT TGGTGGCTAA ATTTAGATAC GAAAGTTCCT GATATTGCAA AGTCTGGTTT 300CACAACTGCT TGGCTGCCTC CGGTGTGTCA GTCATTGGCT CCTGAAGGTT ACCTTCCACA 360GAACCTTTAT TCTCTCAATT CTAAATATGG TTCTGAGGAT CTCTTAAAAG CTTTACTTAA 420TAAGATGAAG CAGTACAAAG TTAGAGCGAT GGCGGACATA GTCATTAACC ACCGTGTTGG 480GACTACTCAA GGGCATGGTG GAATGTACAA CCGCTATGAT GGAATTCCTA TGTCTTGGGA 540TGAACATGCT ATTACATCTT GCACTGGTGG AAGGGGTAAC AAAAGCACTG GAGACAACTT 600TAATGGAGTT CCAAATATAG ATCATACACA ATCCTTTGTT CGGAAAGATC TCATTGACTG 660GATGCGGTGG CTAAGATCCT CTGTTGGCTT CCAAGATTTT CGTTTTGATT TTGCCAAAGG 720TTATGCTTCA AAGTATGTAA AGGAATATAT CGAGGGAGCT GAGCCAATAT TTGCAGTTGG 780AGAATACTGG GACACTTGCA ATTACAAGGG CAGCAATTTG GATTACAACC AAGATAGTCA 840CAGGCAAAGA ATCATCAATT GGATTGATGG CGCGGGACAA CTTTCAACTG CATTCGATTT 900TACAACAAAA GCAGTCCTTC AGGAAGCAGT CAAAGGAGAA TTCTGGCGTT TGCGTGACTC 960TAAGGGGAAG CCCCCAGGAG TTTTAGGATT GTGGCCTTCA AGGGCTGTCA CTTTTATTGA 1020TAATCACGAC ACTGGATCAA CTCAGGCGCA TTGGCCTTTC CCTTCACGTC ATGTTATGGA 1080GGGCTATGCA TACATTCTTA CACACCCAGG GATACCATCA GTTTTCTTTG ACCATTTCTA 1140CGAATGGGAT AATTCCATGC ATGACCAAAT TGTAAAGCTG ATTGCTATTC GGAGGAATCA 1200AGGCATACAC AGCCGTTCAT CTATAAGAAT TCTTGAGGCA CAGCCAAACT TATACGCTGC 1260AACCATTGAT GAAAAGGTTA GCGTGAAGAT TGGGGACGGA TCATGGAGCC CTGCTGGGAA 1320AGAGTGGACT CTCGCGACCA GTGGCCATCG CTATGCAGTC TGGCAGAAGT AATCTTACAG 1380CTATTCCGTT ACTTAATATA TTAGTAGAAA TATATATGTT TTAAACCCGA GCACCTACTT 1440CTAACACTAG ATCCGCCTCT ACAGGCTTGG ATGGAGTGAT GAGTTTTTTT TTCCTGTTCA 1500TTAGACATTG CAACATGGGA TGTATGTTTT GTTAATAAAA GTGTTCTTGA TCAATGCAAT 1560GTAATAAGGG 1570 1570 base pairs nucleic acid double linear DNA (genomic)unknown 19 ACACCACTAG CTTAAAAGTT AAAAAAATGA CTCATAGATC CAACTCCTTGCATTAAAGTT 60 CGACGCTAGC CGAAAAAGGG GACTTGCTCG TTTGTGTCCA ACACCCAAGCTCAATCGTTC 120 CCTGCATATT AGAGTTGATG TTAGGTAATA CCGCGAACTA CTTTCAGTCGTCAGACTAGG 180 TAACCAACAC TATGCGTTAC CTTTCCTCTA GTATAACGTC CGTAAGCTGACCCTTAGAGT 240 ATTTGTACTA ACCACCGATT TAAATCTATG CTTTCAAGGA CTATAACGTTTCAGACCAAA 300 GTGTTGACGA ACCGACGGAG GCCACACAGT CAGTAACCGA GGACTTCCAATGGAAGGTGT 360 CTTGGAAATA AGAGAGTTAA GATTTATACC AAGACTCCTA GAGAATTTTCGAAATGAATT 420 ATTCTACTTC GTCATGTTTC AATCTCGCTA CCGCCTGTAT CAGTAATTGGTGGCACAACC 480 CTGATGAGTT CCCGTACCAC CTTACATGTT GGCGATACTA CCTTAAGGATACAGAACCCT 540 ACTTGTACGA TAATGTAGAA CGTGACCACC TTCCCCATTG TTTTCGTGACCTCTGTTGAA 600 ATTACCTCAA GGTTTATATC TAGTATGTGT TAGGAAACAA GCCTTTCTAGAGTAACTGAC 660 CTACGCCACC GATTCTAGGA GACAACCGAA GGTTCTAAAA GCAAAACTAAAACGGTTTCC 720 AATACGAAGT TTCATACATT TCCTTATATA GCTCCCTCGA CTCGGTTATAAACGTCAACC 780 TCTTATGACC CTGTGAACGT TAATGTTCCC GTCGTTAAAC CTAATGTTGGTTCTATCAGT 840 GTCCGTTTCT TAGTAGTTAA CCTAACTACC GCGCCCTGTT GAAAGTTGACGTAAGCTAAA 900 ATGTTGTTTT CGTCAGGAAG TCCTTCGTCA GTTTCCTCTT AAGACCGCAAACGCACTGAG 960 ATTCCCCTTC GGGGGTCCTC AAAATCCTAA CACCGGAAGT TCCCGACAGTGAAAATAACT 1020 ATTAGTGCTG TGACCTAGTT GAGTCCGCGT AACCGGAAAG GGAAGTGCAGTACAATACCT 1080 CCCGATACGT ATGTAAGAAT GTGTGGGTCC CTATGGTAGT CAAAAGAAACTGGTAAAGAT 1140 GCTTACCCTA TTAAGGTACG TACTGGTTTA ACATTTCGAC TAACGATAAGCCTCCTTAGT 1200 TCCGTATGTG TCGGCAAGTA GATATTCTTA AGAACTCCGT GTCGGTTTGAATATGCGACG 1260 TTGGTAACTA CTTTTCCAAT CGCACTTCTA ACCCCTGCCT AGTACCTCGGGACGACCCTT 1320 TCTCACCTGA GAGCGCTGGT CACCGGTAGC GATACGTCAG ACCGTCTTCATTAGAATGTC 1380 GATAAGGCAA TGAATTATAT AATCATCTTT ATATATACAA AATTTGGGCTCGTGGATGAA 1440 GATTGTGATC TAGGCGGAGA TGTCCGAACC TACCTCACTA CTCAAAAAAAAAGGACAAGT 1500 AATCTGTAAC GTTGTACCCT ACATACAAAA CAATTATTTT CACAAGAACTAGTTACGTTA 1560 CATTATTCCC 1570 20 base pairs nucleic acid single linearcDNA unknown 20 GATAACATTA TGAAGGTGAT 20 21 base pairs nucleic acidsingle linear cDNA unknown 21 GACGGCTGTG GGGTGGAGGA G 21 21 base pairsnucleic acid single linear cDNA unknown 22 CTTGTTATTT GAGTTACTCT C 21 26base pairs nucleic acid single linear cDNA unknown 23 CATAAATTTGTGTCAAATTA TTAAAT 26 23 base pairs nucleic acid single linear cDNAunknown 24 AGGGGTGAAT TGTATAATAT AAT 23 23 base pairs nucleic acidsingle linear cDNA unknown 25 GAAATATTTT TTTTGAAATG GAT 23 23 base pairsnucleic acid single linear cDNA unknown 26 ATTATATTAT ACAATTCACC CCT 2321 base pairs nucleic acid single linear cDNA unknown 27 CGCTTTCCCACCAACGCTGA T 21

What is claimed is:
 1. An isolated α-amylase promoter havingcold-sensitive promoter activity, having a sequence comprising the 5.5Kb EcoRI DNA fragment of Solanum tuberosum from the transformed E. colistrain, DH5alpha-gPAmy 351 (NCIMB Accession Number 40682).
 2. Theα-amylase promoter according to claim 1 operably linked to a gene ofinterest.
 3. An expression vector comprising the α-amylase promoteraccording to claim
 1. 4. A transformation vector comprising theα-amylase promoter according to claim
 1. 5. A construct comprising thepromoter as defined in claim 1 operably linked to a polynucleotideencoding an anti-sense α-amylase polynucleotide.
 6. A transformed plantcell or transformed plant organ comprising the α-amylase promoteraccording to claim
 1. 7. The transformed plant cell or transformed plantorgan of claim 6, wherein said promoter is operably linked to apolynucleotide of interest.
 8. A transgenic plant having a genome thatcomprises the α-amylase promoter according to claim 1 operably linked toa gene of interest.
 9. The transgenic plant according to claim 8,wherein said transgenic plant is a potato plant.
 10. A method ofinducing expression of a polynucleotide of interest in plant cells,comprising the following steps: a) making an expression construct thatcomprises the α-amylase promoter according to claim 1, operably linkedto the polynucleotide of interest; b) introducing said expressionconstruct into cells of a plant to produce cells comprising theexpression construct; and c) exposing said cells comprising theexpression construct to a temperature in the range of from about 0° C.to about 12° C., thereby inducing expression of the polynucleotide ofinterest in the plant cells.
 11. The method of claim 10, wherein saidplant cells are in one or more plant tissues selected from the groupconsisting of: tuber, sprout, root, and stem.
 12. An isolated α-amylasepromoter having tuber-specific activity and having a sequence comprisingSEQ ID NO:
 1. 13. The α-amylase promoter according to claim 12 operablylinked to a gene of interest.
 14. An expression vector comprising theα-amylase promoter according to claim
 12. 15. A transformation vectorcomprising the α-amylase promoter according to claim
 12. 16. Atransformed plant cell or transformed plant organ comprising theα-amylase promoter according to claim
 12. 17. The transformed plant cellor transformed plant organ of claim 16, wherein said promoter isoperably linked to a polynucleotide of interest.
 18. A transgenic planthaving a genome that comprises the α-amylase promoter according to claim12 operably linked to a gene of interest.
 19. A method of expressing apolynucleotide of interest in cells of a plant tuber, comprising thefollowing steps: a) making an expression construct that comprises theα-amylase promoter according to claim 12, operably linked to thepolynucleotide of interest; and b) introducing said expression constructinto cells of a plant tuber to produce cells comprising the expressionconstruct, wherein the polynucleotide of interest is expressed in thecells of the plant tuber.